Hydroxyalkyl starch derivatives

ABSTRACT

The present invention relates to a method of producing a hydroxyalkyl starch derivative comprising reacting hydroxyalkyl starch of formula (I) at its reducing end which is not oxidized prior to said reaction, with a compound of formula (II) R′NH—R″ (II) wherein R 1 -R 2  and R 3  are independently hydrogen or a linear or branched hydroxyalkyl group, and wherein either R′ or R″ or R′ and R″ comprise at least one functional group X capable of being reacted with at least one other compound prior to or after the reaction of (I) and (II), as well as to the hydroxyalkyl starch derivative as such, obtainable by said method, and to a pharmaceutical composition comprising said hydroxyalkyl starch derivative.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part and claims benefit under 35U.S.C. §120 of International Application No. PCT/EP03/08859 having anInternational Filing Date of Aug. 8, 2003, which published in English asInternational Publication Number WO 2004/024777, and which claims thebenefit of priority of European Patent Application No. 02020425.1,having a filing date of Sep. 11, 2002, and U.S. Provisional ApplicationSer. No. 60/409,781 having a filing date of Sep. 11, 2002.

The present invention relates to hydroxyalkyl starch derivates,particularly hydroxyalkyl starch derivatives obtainable by a process inwhich hydroxyalkyl starch is reacted with a primary or secondary aminogroup of a linker compound. According to an especially preferredembodiment, the present invention relates to hydroxyalkyl starchderivatives obtainable by a process according to which hydroxyalkylstarch is reacted with a primary or secondary amino group of a linkercompound and the resulting reaction product is reacted with apolypeptide, preferably with a glycoprotein and especially preferablywith erythropoietin, via at least one other reactive group of the linkercompound. A hydroxyalkyl starch which is especially preferred ishydroxyethyl starch. According to the present invention, thehydroxyalkyl starch and preferably the hydroxylethyl starch is reactedwith the linker compound at its reducing end which is not oxidized priorto said reaction.

Hydroxyethyl starch (HES) is a derivative of naturally occurringamylopectin and is degraded by alpha-amylase in the body. HES is asubstituted derivative of the carbohydrate polymer amylopectin, which ispresent in corn starch at a concentration of up to 95% by weight. HESexhibits advantageous biological properties and is used as a bloodvolume replacement agent and in hemodilution therapy in the clinics(Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278; andWeidler et al., 1991, Arzneim.-Forschung/Drug Res., 41, 494-498).

Amylopectin consists of glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physical-chemical propertiesof this molecule are mainly determined by the type of glycosidic bonds.Due to the nicked alpha-1,4-glycosidic bond, helical structures withabout six glucose-monomers per turn are produced. The physico-chemicalas well as the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

Some ways of producing a hydroxyethyl starch derivative are described inthe art.

DE 26 16 086 discloses the conjugation of hemoglobin to hydroxyethylstarch wherein, in a first step, a cross-linking agent, e.g. bromocyane,is bound to hydroxyethyl starch and subsequently hemoglobin is linked tothe intermediate product.

One important field in which HES is used is the stabilisation ofpolypeptides which are applied, e.g., to the circulatory system in orderto obtain a particular physiological effect. One specific example ofthese polypeptides is erythropoietin, an acid glycoprotein ofapproximately 34,000 kD which is essential in regulating the level ofred blood cells in the circulation.

A well-known problem with the application of polypeptides and enzymes isthat these proteins often exhibit an unsatisfactory stability.Especially erythropoietin has a relatively short plasma half live(Spivak and Hogans, 1989, Blood 73, 90; McMahon et al., 1990, Blood 76,1718). This means that therapeutic plasma levels are rapidly lost andrepeated intravenous administrations must be carried out. Furthermore,in certain circumstances an immune response against the peptides isobserved.

It is generally accepted that the stability of polypeptides can beimproved and the immune response against these polypeptides is reducedwhen the polypeptides are coupled to polymeric molecules. WO 94/28024discloses that physiologically active polypeptides modified withpolyethyleneglycol (PEG) exhibit reduced immunogenicity and antigenicityand circulate in the bloodstream considerably longer than unconjugatedproteins, i.e. have a longer clearance rate. However, PEG-drugconjugates exhibit several disadvantages, e.g. they do not exhibit anatural structure which can be recognized by elements of in vivodegradation pathways. Therefore, apart from PEG-conjugates, otherconjugates and protein polymerates have been produced. A plurality ofmethods for the cross-linking of different proteins and macromoleculessuch as polymerase have been described in the literature (see e.g. Wong,Chemistry of protein conjugation and cross-liking, 1993, CRCS, Inc.).

The HES-drug conjugates disclosed in the art suffer from thedisadvantage that HES is not conjugated site-specifically to the drug.Consequently, the conjugation results in a very heterogenous producthaving many components that may be inactive due to the destruction ofthe 3-dimensional structure during the conjugation step. Therefore,there is a need for further improved HES-polypeptides conjugates withimproved stability and/or bioactivity.

One method of producing these conjugates uses, as starting material, anoxidized form of HES which is reacted with a crosslinking compoundwherein the resulting product is reacted with a polypeptide or furthermodified and subsequently reacted with a polypeptide. It is a majordisadvantage of this method that in a first step, the original HES hasto be selectively oxidized, generally at its reducing end, by oxidizingthe terminal aldehyde group and/or hemiacetale group to a lactone, thusrendering the overall process more difficult and expensive.

WO 02/08079 A2 discloses compounds comprising a conjugate of an activeagent and a hydroxyalkyl starch wherein active agent and hydroxyalyklstarch are either linked directly or via a linker compound. As far asthe direct linkage is concerned, the reaction of active agent andhydroxyalkyl starch is carried out in an aqueous medium which comprisesat least 10 wt.-% of water. No examples are given which are directed toa hydroxyalkyl starch derivative which is produced by reactinghydroxyalkyl starch at its reducing end with a crosslinking compoundcomprising the structure unit —NH— in an aqueous medium. All examplesare directed to hydroxyalkyl starch which is oxidized prior to a furtherreaction, the specific teaching of WO 02/08079 A2 thus having theaformentioned disadvantages.

Therefore, it is an object of the present invention to provide a methodof producing a hydroxyalkyl starch derivative which allows for reactinghydroxyalkyl starch at its reducing end with a suitable compound whereinthe reducing end of the starch is not oxidized prior to the reaction.

It is a further object of the present invention to provide a method ofproducing a hydroxyalkyl starch derivative which allows for reactinghydroxyalkyl starch at its reducing end with a suitable compound whereinthe reducing end of the starch is not oxidized prior to the reaction,said method being further characterized in that the reaction product ofthe reaction of hydroxyalkyl starch at its reducing end with a suitablecompound is further reacted with at least one further compound.

It is a still further object of the present invention to provide amethod as described above wherein the at least one further compound is apolypeptide, preferably a protein, more preferably erythropoietin.

It is yet another object of the present invention to provide ahydroxyalkyl starch derivative which is obtainable by a method asdescribed above which comprises reacting hydroxyalkyl starch at itsreducing end with a suitable compound wherein the reducing end of thestarch is not oxidized prior to the reaction.

Therefore, the present invention relates to a method of producing ahydroxyalkyl starch derivative comprising reacting hydroxyalkyl starch(HAS) of formula (I)

at its reducing end which is not oxidized prior to said reaction, with acompound of formula (II)R′—NH—R″  (II)wherein R₁, R₂ and R₃ are independently hydrogen or a linear or branchedhydroxyalkyl group, and wherein either R′ or R″ or R′ and R″ comprise atleast one functional group X capable of being reacted with at least oneother compound prior to or after the reaction of (I) and (II).

In the context of the present invention, the term “hydroxyalkyl starch”(HAS) refers to a starch derivative which has been substituted by atleast one hydroxyalkyl group. Therefore, the term hydroxyalkyl starch asused in the present invention is not limited to compounds where theterminal carbohydrate moiety comprises hydroxyalkyl groups R₁, R₂,and/or R₃ as depicted, for the sake of brevity, in formula (I), but alsorefers to compounds in which at least one hydroxy group presentanywhere, either in the terminal carbohydrate moiety and/or in theremaining part of the starch molecule, HAS′, is substituted by ahydroxyalkyl group R₁, R₂, or R₃.

In this context, the alkyl group may be a linear or branched alkyl groupwhich may be suitably substituted. Preferably, the hydroxyalkyl groupcontains 1 to 10 carbon atoms, more preferably from 1 to 0.6 carbonatoms, more preferably from 1 to 4 carbon atoms, and even morepreferably 2-4 carbon atoms. “Hydroxyalkyl starch” therefore preferablycomprises hydroxyethyl starch, hydroxypropyl starch and hydroxybutylstarch, wherein hydroxyethyl starch and hydroxypropyl starch areparticularly preferred.

Hydroxyalkyl starch comprising two or more different hydroxyalkyl groupsare also possible.

The at least one hydroxyalkyl group comprised in HAS may contain two ormore hydroxy groups. According to a preferred embodiment, the at leastone hydroxyalkyl group comprised HAS contains one hydroxy group.

The expression “hydroxyalkyl starch” also includes derivatives whereinthe alkyl group is mono- or polysubstituted. In this context, it ispreferred that the alkyl group is substituted with a halogen, especiallyfluorine, or with an aryl group, provided that the HAS remains solublein water. Furthermore, the terminal hydroxy group a of hydroxyalkylgroup may be esterified or etherified.

Furthermore, instead of alkyl, also linear or branched substituted orunsubstituted alkene groups may be used.

Hydroxyalkyl starch is an ether derivative of starch. Besides of saidether derivatives, also other starch derivatives can be used in thecontext of the present invention. For example, derivatives are usefulwhich comprise esterified hydroxy groups. These derivatives may be e.g.derivatives of unsubstituted mono- or dicarboxylic acids with 2-12carbon atoms or of substituted derivatives thereof. Especially usefulare derivatives of unsubstituted monocarboxylic acids with 2-6 carbonatoms, especially derivatives of acetic acid. In this context, acetylstarch, butyl starch and propyl starch are preferred.

Furthermore, derivatives of unsubstituted dicarboxylic acids with 2-6carbon atoms are preferred.

In the case of derivatives of dicarboxylic acids, it is useful that thesecond carboxy group of the dicarboxylic acid is also esterified.Furthermore, derivatives of monoalkyl esters of dicarboxylic acids arealso suitable in the context of the present invention.

For the substituted mono- or dicarboxylic acids, the substitute groupsmay be preferably the same as mentioned above for substituted alkylresidues.

Techniques for the esterification of starch are known in the art (seee.g. Klemm D. et al, Comprehensive Cellulose Chemistry Vol. 2, 1998,Whiley-VCH, Weinheim, N.Y., especially chapter 4.4, Esterification ofCellulose (ISBN 3-527-29489-9).

Hydroxyethyl starch (HES) is most preferred for all embodiments of thepresent invention.

Therefore, the present invention also relates to a method as describedabove wherein the hydroxyalkyl starch is hydroxyethyl starch.

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. There are two possibilities of describing thesubstitution degree:

-   1. The substitution degree can be described relatively to the    portion of substituted glucose monomers with respect to all glucose    moieties (DS).-   2. The substitution degree can be described as the “molar    substitution” (MS), wherein the number of hydroxyethyl groups per    glucose moiety are described.

HES solutions are present as polydisperse compositions, wherein eachmolecule differs from the other with respect to the polymerisationdegree, the number and pattern of branching sites, and the substitutionpattern. HES is therefore a mixture of compounds with differentmolecular weight. Consequently, a particular HES solution is determinedby average molecular weight with the help of statistical means. In thiscontext, M_(n) is calculated as the arithmetic mean depending on thenumber of molecules. Alternatively, M_(w), the weight mean, represents aunit which depends on the mass of the HES.

In the context of the present invention, hydroxyethyl starch may have amean molecular weight (weight mean) of from 1 to 300 kDa, wherein a meanmolecular weight of from 5 to 100 kDa is more preferred. Hydroxyethylstarch can further exhibit a molar degree of substitution of from 0.1 to0.8 and a ratio between C₂:C₆ substitution in the range of from 2 to 20with respect to the hydroxyethyl groups.

As far as the residues R₁, R₂ and R₃ according to formula (I) areconcerned there are no specific limitations given that compound (I)remains capable of being reacted with a compound according to formula(II). According to a preferred embodiment, R₁, R₂ and R₃ areindependently hydrogen or a hydroxyalkyl group, a hydroxyaryl group, ahydroxyaralkly group or a hydroxyalkarly group having of from 1 to 10carbon atoms. Hydrogen and hydroxyalkyl groups having of from 1 to 6carbon atoms are preferred. The alkyl, aryl, aralkyl and/or alkarylgroup may be linear or branched and suitably substituted.

Therefore, the present invention also related to a method as describedabove wherein R₁, R₂ and R₃ are independently hydrogen or a linear orbranched hydroxyalkyl group with from 1 to 6 carbon atoms.

Thus, R₁, R₂ and R₃ may be hydroxyhexyl, hydroxypentyl, hydroxybutyl,hydroxypropyl such as 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,1-hydroxyisopropyl, 2-hydroxyisopropyl, hydroxyethyl such as1-hydroxyethyl, 2-hydroxyethyl, or hydroxymethyl. Hydrogen andhydroxyethyl groups are preferred, hydrogen and the 2-hydroxyethyl groupbeing especially preferred.

Therefore, the present invention also relates to a method as describedabove wherein R₁, R₂ and R₃ are independently hydrogen or a2-hydroxyethyl group.

According to the present invention, hydroxyalkyl starch is reacted witha compound of formula (II) wherein compound (II) may be reacted withanother compound prior to the reaction with compound (I), to give ahydroxyalkyl starch derivative. As to compound (II), there are nospecific limitations if compound (II) is capable of being reacted viathe NH group bridging R′ and R″ with compound (I) at its reducing endwhich is not oxidized, to give a hydroxyalkyl starch derivative.

Preferred residues R′ of compound (II) are hydrogen and alkyl,cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylarylresidues where cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl orcycloalkylaryl residues may be linked directly to the NH group bridgingR′ and R″ of compound (II) or, according to another embodiment, may belinked by an oxygen bridge to the NH group bridging R′ and R″ ofcompound (II). The alkyl, aryl, aralkyl or alkaryl residues may besuitably substituted. As preferred substituents, halogenes such as F, Clor Br may be mentioned. Especially preferred residues R′ are hydrogen,alkyl and alkoxy groups, and even more preferred are hydrogen andunsubstituted alkyl and alkoxy groups.

Therefore, the present invention also relates to a method as describedabove wherein R′ is hydrogen or a linear or branched alkyl or alkoxygroup.

Among the alkyl and alkoxy groups, groups with 1, 2, 3, 4, 5, or 6 Catoms are preferred. More preferred are methyl, ethyl, propyl,isopropyl, methoxy, ethoxy, propoxy, and isopropoxy groups. Especiallypreferred are methyl, ethyl, methoxy, ethoxy, and particular preferenceis given to methyl or methoxy.

Therefore, the present invention also relates to a method as describedabove wherein R′ is hydrogen or a methyl or a methoxy group.

Apart from the functional group X, R″ may comprise at least oneadditional functional group W. This at least one additional functionalgroup W generally may be anywhere in R″. Preferably, W is directlylinked to the NH group R′ is linked to.

In general, there are no specific limitations regarding functional groupW given that compound (I) is capable of being reacted with compound(II). In preferred embodiments, functional group W comprises thestructure unit —NH— and/or the structure unit —(C=G)- where G is O or S,and/or the structure unit —SO₂—. According to more preferredembodiments, the functional group W is selected from the groupconsisting of

where, if G is present twice, it is independently O or S.

According to preferred embodiments of the present invention where R′ isH and W is linked directly to the NH group bridging R′ and R″, R′ andthe NH group bridging R′ and R″ form, together with W, the one offollowing groups:

As far as the at least one functional group X which is comprised in R′and/or R″, preferably in R″, no specific limitations exist. In general,all functional groups are possible which allow the reaction with atleast one further compound.

As far as this reaction with a further compound is concerned, all kindsof interactions of the at least one functional group with the at leastone further compound are possible. Among others, reactions of the atleast one functional group X with a further compound are possible whichlead to a covalent linkage, a ionic linkage and/or a van-der-Waalslinkage, the covalent linkage being especially preferred.

Among others, the following functional groups X are to be mentioned:

-   -   C—C-double bonds or C—C-triple bonds or aromatic C—C-bonds;    -   the thio group or the hydroxy groups;    -   alkyl sulfonic acid hydrazide, aryl sulfonic acid hydrazide;    -   1,2-dioles;    -   1,2-aminoalcohols;    -   the amino group —NH₂ or derivatives of the amino groups        comprising the structure unit —NH— such as aminoalkyl groups,        aminoaryl group, aminoaralkyl groups, or alkarlyamino groups;    -   the hydroxylamino group —O—NH₂, or derivatives of the        hydroxylamino group comprising the structure unit —O—NH—, such        as hydroxylalkylamino groups, hydroxylarylamino groups,        hydroxylaralkylamino groups, or hydroxalalkarylamino groups;    -   alkoxyamino groups, aryloxyamino groups, aralkyloxyamino groups,        or alkaryloxyamino groups, each comprising the structure unit        —NH—O—;    -   residues having a carbonyl group, —Q—C(=G)—M, wherein G is O or        S, and M is, for example,        -   —OH or —SH;        -   an alkoxy group, an aryloxy group, an aralkyloxy group, or            an alkaryloxy group;        -   an alkylthio group, an arylthio group, an aralkylthio group,            or an alkarylthio group;        -   an alkylcarbonyloxy group, an arylcarbonyloxy group, an            aralkylcarbonyloxy group, an alkarylcarbonyloxy group;        -   activated esters such as esters of hydroxylamines having            imid structure such as N-hydroxysuccinimide or having a            structure unit O—N where N is part of a heteroaryl compound            or, with G=O and Q absent, such as aryloxy compounds with a            substituted aryl residue such as pentafluorophenyl,            paranitrophenyl or trochlorophenyl;    -   wherein Q is absent or NH or a heteroatom such as S or O;    -   —NH—NH₂, or —NH—NH—;    -   —NO₂;    -   the nitril group;    -   carbonyl groups such as the aldehyde group or the keto group;    -   the carboxy group;    -   the —N═C—O group or the —N═C═S group;    -   vinyl halide groups such as the vinyl iodide or the vinyl        bromide group or vinyltriflate;    -   —C≡C—H;    -   —(C═NH₂Cl)—OAlkyl    -   groups —(C═O)—CH₂-Hal wherein Hal is Cl, Br, or I;    -   —CH═CH—SO₂—;    -   a disulfide group comprising the structure —S—S—;    -   the group

-   -   the group

Among these groups, the thio group, the amino group, the hydroxylaminogroup, the alkoxyamino groups and the following groups are especiallypreferred:

Therefore, the present invention also relates to a method as describedabove wherein the at least one functional group X is selected from thegroup consisting of —SH, —NH₂, —O—NH₂, —NH—O-alkyl, —(C=G)—NH—NH₂,—G—(C=G)—NH—NH₂, —NH—(C=G)—NH—NH₂, and —SO₂—NH—NH₂ where G is O or Sand, if G is present twice, it is independently O or S.

As far as the alkoxyamino groups are concerned, particular preference isgiven to the propoxyamino group, the ethoxyamino group and themethoxyamino group, the methoxyamino group —NH—O—CH₃ being especiallypreferred.

According to yet another aspect of the present invention, the at leastone functional group X may be a group which is not capable of reactingdirectly with a given further compound but which may be chemicallymodified in order to be capable of reacting in the desired way. Thismodification of the functional group X comprised in compound (II) may becarried out either prior to the reaction of compound (II) with compound(I) or after the reaction of compound (II) with compound (I). Ifcompound (II) comprises at least two, optionally chemically different,functional groups X, it is possible to modify at least one functionalgroup X prior to the reaction of compound (II) with compound (I) and atleast one functional group X after the reaction of compound (II) withcompound (I).

As an example of a functional group X to be modified prior to thereaction with a further compound, a 1,2-amino alcohol or a 1,2-diol maybe mentioned which is modified, e.g., by oxidation to form an aldehyd ora keto group.

Another example for a functional group X to be modified prior to thereaction with a further compound is a —NH₂ group which is modified bythe reaction with, e.g., a compound according to the following formula

to give a structure of the following formula

which is, e.g., reactive towards a thio group.

Another example for a functional group X to be modified prior to thereaction with a further compound is a —NH₂ group which is modified bythe reaction with, e.g., a compound according to the following formula

to give a structure of the following formula

which is, e.g., reactive towards a thio group.

The at least one functional group X may be linked directly to the NHgroup bridging R′ and R″. Thus, according to one embodiment of thepresent invention, the functional group X is equivalent to R″. Specificexamples of compounds where X is directly linked to the NH groupbridging R′ and R″ are, among others,

Another specific example of a such a compound which is also comprised inthe present invention is NH₃.

According to another embodiment of the present invention, the NH groupbridging R′ and R″ may be separated from the at least one functionalgroup X by a linear or branched alkyl or cycloalkyl or aryl or aralkylor arylcycloalkyl or alkaryl or cycloalkylaryl group, wherein thesegroups may comprise at least one heteroatom such as N, O, S, and whereinthese groups may be suitably substituted. The size of the groupseparating NH, bridging R′ and R″, and the at least one functional groupX may be adapted to the specific needs. Generally, the separating grouphas generally from 1 to 60, preferably from 1 to 40, more preferablyfrom 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6and especially preferably from 1 to 4 carbon atoms. If heteroatoms arepresent, the separating group comprises generally from 1 to 20,preferably from 1 to 8 and especially preferably from 1 to 4heteroatoms. According to particularly preferred embodiments of thepresent invention, the separating group comprises 1 to 4 oxygen atoms.The separating group may comprise an optionally branched alkyl chain oran aryl group or a cycloalkyl group having, e.g., from 5 to 7 carbonatoms, or be a aralkyl group, an alkaryl group where the alkyl part maybe a linear and/or cyclic alkyl group. According to an even morepreferred embodiment, the separating group is an alkyl chain of from 1to 20, preferably from 1 to 8, more preferably from 1 to 6, morepreferably from 1 to 4 and especially preferably from 2 to 4 carbonatoms. In case heteroatoms are present, a chain comprising 1 to 4 oxygenatoms is particularly preferred.

Specific examples of compounds (II) where X is separated from the NHgroup bridging R′ and R″ are, among others,

The group separating NH, bridging R′ and R″, and the at least onefunctional group X may be suitably substituted. Preferred substituentsare, e.g, halides such as F, Cl, Br or I.

The group separating NH, bridging R′ and R″, and the at least onefunctional group X may comprise one or more cleavage sites such as

which allow for an easy cleavage of a resulting compound at apre-determined site.

According to an especially preferred embodiment of the presentinvention, the compound (II) is O-[2-2-aminooxy-ethoxy)-ethyl]-hydroxylamine

or carbohydrazide

Therefore, the present invention also relates to a method as describedabove wherein compound (II) is O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine or carbohydrazide.

In case compound (II) comprises one or more chiral centers, compound(II) may be present in R conformation or in S conformation or as racemiccompound with respect to each chiral center.

As described above, compound (I) may be reacted with compound (II) assuch or with compound (II) which has been reacted with at least onefurther compound prior to the reaction with compound (I).

The reaction of compound (I) with compound (II) as such may be carriedout in at least one suitable solvent. The respective solvent or mixtureof two or more solvents may be adapted to the specific needs of thereaction conditions and the chemical nature of compounds (I) and (II).According to an especially preferred embodiment of the presentinvention, water is used as solvent, either alone or in combination withat least one other solvent. As at least one other solvent, DMSO, DMF,methanol and ethanol may be mentioned. Preferred solvents other thanwater are DMSO, DMF, methanol and ethanol.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of compound (I) with compound (II) is carriedout in an aqueous system.

The term “aqueous system” as used in the context of the presentinvention refers to a solvent or a mixture of solvents comprising waterin the range of from at least 10% per weight, preferably at least 50%per weight, more preferably at least 80% per weight, even morepreferably at least 90% per weight or up to 100% per weight, based onthe weight of the solvents involved. The preferred reaction medium iswater.

As far as the temperatures which are applied during the reaction areconcerned, no specific limitations exist given that the reaction resultsin the desired hydroxyalkyl starch derivative.

In case compound (I) is reacted with compound (II), compound (II) beinga hydroxylamine or a hydrazide, the temperature is preferably in therange of from 5 to 45° C., more preferably in the range of from 10 to30° C. and especially preferably in the range of from 15 to 25° C.

In case compound (I) is reacted with compound (II), said reaction beinga reductive amination, the temperature is preferably in the range of upto 100° C., more preferably in the range of from 20 to 95° C., morepreferably in the range of from 25 to 90° C., more preferably in therange of from 70 to 90° C. and especially preferably in the range offrom 75 to 85° C.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of compound (I) and compound (II), compound(II) being a hydroxylamine or a hydrazide, is carried out at atemperature of from 5 to 45° C.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of compound (I) and compound (II), saidreaction being a reductive amination, is carried out at a temperature offrom 25 to 90° C.

During the course of the reaction the temperature may be varied,preferably in the above-given ranges, or held essentially constant.

The reaction time for the reaction of compound (I) with (II) may beadapted to the specific needs and is generally in the range of from 1 hto 7 d.

In case compound (II) is a hydroxylamine or a hydrazide, the reactiontime is preferably in the range of from 1 h to 3 d and more preferablyof from 2 h to 48 h.

In case the reaction of compound (I) and compound (II) is a reductiveamination, the reaction time is preferably in the range of from 2 h to 7d.

The pH value for the reaction of compound (I) with (II) may be adaptedto the specific needs such as the chemical nature of the reactants.

In case compound (II) is a hydroxylamine or a hydrazide, the pH value ispreferably in the range of from 4.5 to 6.5.

In case the reaction of compound (I) and compound (II) is a reductiveamination, the pH value is preferably in the range of from 8 to 12.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of compound (I) and compound (II), compound(II) being a hydroxylamine or a hydrazide, is carried out at a pH offrom 4.5 to 6.5.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of compound (I) and compound (II), saidreaction being a reductive amination, is carried out at a pH of from 8to 12.

Specific examples of above mentioned reaction conditions are, e.g., areaction temperature of about 25° C. and a pH of about 5.5 in casecompound is a hydroxylamine, and a reaction temperature of about 80° C.and a pH of about 11 in case the reaction of compound (I) and compound(II) is a reductive amination.

The suitable pH value of the reaction mixture may be adjusted by addingat least one suitable buffer. Among the preferred buffers, sodiumacetate buffer, phosphate or borate buffers may be mentioned.

According to a preferred embodiment of the present invention, thereaction product resulting from the reaction of compound (I) withcompound (II) is reacted with at least one further compound via the atleast one functional group X.

If necessary, the at least one functional group X may be protected withat least one suitable protecting group prior to the reaction of compound(I) with compound (II). In this respect, all conceivable protectinggroups are possible which prevent the protected compound (II) fromreacting with compound (I) via the at least one functional group X.Hence, the protecting group may be chosen depending from the chemicalnature of the functional group X to be protected, from, e.g., thesolvent the reaction is carried out in or the pH of the reactionmixture. Preferred protecting groups are, among others, thebenzyloxycarbonyl group, the tert-butoxycarbonyl group, themethoxyphenyl group, the 2,4-dimethoxyphenyl group, triarly methylgroups, trityl, the monomethoxytrityl group, the dimethoxytrityl group,the monomethyltrityl group, the dimethyltrityl group, the trifluoracetylgroup, phthalimin compounds, 2-(trialkylslyl)ethoxy carbonyl compounds,Fmoc, the tert-butyl group, or trialkyl silyl groups.

If two or more different functional groups X are present in compound(II), at least one group may be protected whereas at least one othergroup may be left unprotected.

After the reaction of compound (I) with compound (II), the at least oneprotecting group may be left in the reaction product or removed bysuitable methods such as conventional methods known to the personskilled, in the art. If two different functional groups X are protectedby suitable protecting groups, it is possible to remove at least oneprotecting group so as to make at least one functional group X availablefor further reaction with at least one further compound, and leave atleast one other functional group protected until the reaction product ofcompound (I) with compound (II) is reacted with the further compound.Afterwards, the protecting group of the functional group still protectedmay be removed to make the remaining functional group X available forreaction with yet a further compound.

The use of at least one protecting group may be important for preventingthe reaction from resulting in a hydroxyalkyl starch derivativeconsisting of a compound (II) which has been reacted with two or morecompounds (I), i.e. a multiple HAS substituted compound (II). The sameresult, however, may be achieved by reacting compound (I) with an excessof compound (II). If an excess amount of compound (II) is used in theprocess of the present invention, the molar ratio of compound (II) tocompound (I) is preferably in the range of from 2 to 100.

Once the reaction product of the reaction of compound (I) with compound(II) is formed, it may be isolated from the reaction mixture by at leastone suitable method. If necessary, the reaction product may beprecipitated prior to the isolation by at least one suitable method.

If the reaction product is precipitated first, it is possible, e.g., tocontact the reaction mixture with at least one solvent or solventmixture other than the solvent or solvent mixture present in thereaction mixture at suitable temperatures. According to a particularlypreferred embodiment of the present invention where water is used assolvent, the reaction mixture is contacted with a mixture of ethanol andacetone, preferably a 1:1 mixture, indicating equal volumes of saidcompounds, at a temperature, preferably in the range of from −20 to +50°C. and especially preferably in the range of from 0 to 25° C.

Isolation of the reaction product may be carried out by a suitableprocess which may comprise one or more steps. According to a preferredembodiment of the present invention, the reaction product is firstseparated off the reaction mixture or the mixture of the reactionmixture with, e.g., the ethanol-acetone mixture, by a suitable methodsuch as centrifugation or filtration. In a second step, the separatedreaction product may be subjected to a further treatment such as anafter-treatment like dialysis, centrifugal filtration or pressurefiltration, ion exchange chromatography, HPLC, MPLC, gel filtrationand/or lyophilisation. According to an even more preferred embodiment,the separated reaction product is first dialysed, preferably againstwater, and then lyophilized until the solvent content of the reactionproduct is sufficiently low according to the desired specifications ofthe product. Lyophilisation may be carried out at temperature of from 20to 35° C., preferably of from 25 to 30° C.

The thus isolated reaction product of compound (I) and compound (II) maybe further reacted with at least one other compound via at least onefunctional group X comprised in said reaction product.

Depending on the chemical nature of the functional group X, everyconceivable compound capable of forming a chemical linkage with thisgroup X may be used. For this reaction, one or more suitable solventsmay be used, and all reaction parameters such as the temperature duringthe reaction, the reaction time, the ratios of the reactants or the pHvalue of the reaction mixture may be adapted to the specific needs.

According to a particularly preferred embodiment of the presentinvention, the at least one compound capable of forming a chemicallinkage with the at least one functional group X is a polypeptide or amixture of at least two different polypeptides.

Therefore, the present invention also relates to a method as describedabove wherein the reaction product of compound (I) and compound (II) isreacted with a polypeptide via the functional group X comprised incompound (II).

According to another particularly preferred embodiment of the presentinvention, the at least one further compound capable of forming achemical linkage with the at least one functional group X is acrosslinking compound which is capable of forming a first chemicallinkage with the at least one functional group X of the reaction productof compound (I) and compound (II), and a second chemical linkage withsecond further compound.

According to an even more preferred embodiment of the present invention,the second further compound is a polypeptide or a mixture of at leasttwo different polypeptides.

In the context of this embodiment of the present invention, it ispossible to react the reaction product of compound (I) and compound(II), the first hydroxyalkyl starch derivative, with the crosslinkingcompound to give a second hydroxyalykl starch derivative. This secondhydroxyalykl starch derivative may be subsequently reacted with thesecond further compound, preferably a polypeptide, to give a thirdhydroxyalykl starch derivative.

It is, however, also possible to react the reaction product of compound(I) and compound (II), the first hydroxyalykl starch derivative, with areaction product of the crosslinking compound with the second furthercompound, preferably a polypeptide.

Therefore, the present invention also relates to a method as describedabove wherein the reaction product of compounds (I) and (II) is reactedwith a further compound, said further compound being a crosslinkingcompound, via reaction of a functional group V comprised in thecrosslinking compound and a functional group X comprised in the reactionproduct of compounds (I) and (II).

Therefore, the present invention also relates to a method as describedabove wherein the reaction product of compounds (I) and (II) is reactedwith a further compound, said further compound being a crosslinkingcompound, via reaction of a functional group V comprised in thecrosslinking compound and a functional group X comprised in the reactionproduct of compounds (I) and (II), said crosslinking compound havingbeen reacted with a second further compound prior to the reaction withthe reaction product of compounds (I) and (II).

Therefore, the present invention also relates to a method as describedabove wherein the second further compound is a polypeptide, preferablyerythropoietin, which is reacted with the crosslinking compound viareaction of a functional group X, comprised in the crosslinkingcompound.

Therefore, the present invention also relates to a method as describedabove wherein compound (II) is reacted with a first further compound,preferably a crosslinking compound, to give a first reaction product,said first reaction product is reacted with a second further compound togive a second reaction product, and said second reaction product isreacted with compound (I).

Therefore, the present invention also relates to a method as describedabove wherein a first further compound, preferably a crosslinkingcompound, is reacted with a second further compound, preferably apolypeptide, to give a first reaction product, said first reactionproduct is reacted with compound (II) to give a second reaction product,and said second reaction product is reacted with compound (I) to givethe hydroxyalkyl starch derivative.

According to especially preferred embodiments of the present invention,the crosslinking compounds are used to form a chemical bridge betweencompound (II) or the reaction product of compounds (I) and (II), and asecond further compound wherein the functional group of the secondfurther compound which reacts with the crosslinking compound is a —SHgroup or an aldehyde group or a keto group, and the functional group ofcompound (II) or the reaction product of compounds (I) and (II) whichreacts with the crosslinking compound is a group comprising thestructure —NH—, particularly preferably —NH₂.

In the context of the present invention, the term “crosslinkingcompound” relates to chemical compounds which are capable of forming alinkage between compound (II) or the reaction product of compounds (I)and (II), and at least one given second further compound. Depending onthe chemical nature of the second further compound, the crosslinkingcompound comprises at least one functional group V capable of beingreacted with the functional group X comprised in compound (II) or thereaction product of compounds (I) and (II), and at least one furtherfunctional group which is capable of forming a chemical linkage with thesecond further compound. This at least one further functional groupcomprised in the crosslinking compound may be a functional group of thetype discussed above with regard to the functional group X.

The crosslinking compound may be used to enlarge the length of theoverall chemical bridge between compound (I) and the second furthercompound, preferably a polypeptide, and/or to influence the chemicalnature of the resulting reaction product, either with or without thesecond further compound, and/or provide the possibility to form alinkage between several second further compounds and the reactionproduct of compound (I), (II) and the crosslinking compound, and/or tochemically modify the functional group X comprised in the reactionproduct of compound (I) and (II) so as to render said reaction productcapable of reacting with a given further compound.

Thus, embodiments of the present invention which are discussed above andwhich relate to the chemical modification of the functional group Xbeing a —NH₂ group, with a further compound, e.g.

in order to provide the possibility for the reaction with an —SH groupcomprised in a second further compound, preferably a polypeptide, arespecific examples of reacting the reaction product of compounds (I) and(II) with a crosslinking compound.

According to a preferred embodiment of the present invention, thefunctional group V may be a functional group of the type discussed aboveas group X.

According to another preferred embodiment, either functional group X orfunctional group V is a thio group and functional group V or functionalgroup X is preferably selected from the group consisting of

wherein Hal is Cl, Br, or I, preferably Br or I.

According to yet another preferred embodiment, either functional group Xor functional group V is selected from the group consisting of anactivated ester as described above or a carboxy group which isoptionally transformed into an activated ester. In this particular case,the functional group V or the functional group X, respectively,comprises the chemical structure —NH—.

Therefore, the crosslinking compound is a compound having at least twofunctional groups which are the same or different. In the case of twofunctional group, the crosslinking compound may be homo-bifuncational orhetero-bifunctional. A homobifunctional crosslinking compound, e.g.,provides the possibility to form a bridge between the reaction productof compounds (I) with (II) and a second further compound, the reactionproduct and the further compound having the same type of functionalgroups. A hetero-bifunctional crosslinking compound, e.g., provides thepossibility to form a bridge between the reaction product of compounds(I) with (II) and a second further compound, the reaction product andthe further compound having functional groups which are not capable ofreacting with each other.

The at least two functional groups of the crosslinking compound may belinked directly together or may be separated by a linear or branchedalkyl or cycloalkyl or aryl or aralkyl or arylcycloalkyl or alkaryl orcycloalkylaryl group, wherein these groups may comprise at least oneheteroatom such as N, O, S, and wherein these groups may be suitablysubstituted. The length of the group separating the at least twofunctional groups of the crosslinking compound may be adapted to thespecific needs. Generally, the separating group has from 1 to 60,preferably from 1 to 40, more preferably from 1 to 20, more preferablyfrom 1 to 10, more preferably from 5 to 10 carbon atoms. If heteroatomsare present, the separating group comprises generally from 1 to 20,preferably from 1 to 8 and especially preferably from 1 to 4heteroatoms. According to an even more preferred embodiment, theseparating group is an alkyl or aralkyl chain of from 1 to 20 carbonatoms. Moreover, the crosslinking compound may further comprise at leastone cleavage site as discussed above with regard to compound (II).

Other examples of crosslinking compounds which are to be mentioned inthe context of the present invention may be categorized, e.g., accordingto the following list:

Type of Functional group, capable of cross- being reacted with a secondlinking further compound, preferably compound a polypeptide Functionalgroup V A Hydrazide (aldehyde-reactive) Maleimido (SH-reactive BHydrazide (aldeyde-reactive) Pydridydithio (SH- reactive) C Iodoalkyl(SH-reactive) N-succinimide ester (amine-reactive) D Bromoalkyl(SH-reactive) N-succinimide ester (amine-reactive) E Maleimido(SH-reactive) N-succinimide ester (amine-reactive) F Pydridyldithio(SH-reactive) N-succinimide ester (amine-reactive) G Vinylsulfone(SH-reactive) N-succinimide ester (amine-reactive)

In Table 1 at the end of the present description, some preferredexamples of crosslinking compounds are listed.

In case the at least one further compound, e.g. the crosslinkingcompound, comprises one or more chiral centers, the at least one furthercompound may be present in R conformation or in S conformation or asracemic compound with respect to each chiral center.

The term “polypeptide” as used in the context of the present inventionrefers to a compound which comprises at least 2 amino acids which arelinked via a peptide bond, i.e. a bond with structure —(C═O)—NH—. Thepolypeptide may be a naturally occurring compound or a polypeptide whichdoes not occur naturally, the latter comprising naturally occurringamino acids and/or at least one amino acid which does not naturallyoccur. The backbone of the polypeptide, the polypeptide chain, may befurther substituted with at least one suitable substituent thus havingat least one sidechain. The at least one functional group Y may be partof the polypeptide backbone or of at least one substituent of thebackbone wherein embodiments are possible comprising at least onefunctional group being part of the polypeptide backbone and at least onefunctional group being part of at least one substituent of thepolypeptide backbone.

As far as the polypeptide is concerned, there exist no restrictions,given that the polypeptide comprises at least one functional group Y.Said functional group Y may be linked directly to the polypeptidebackbone or be part of a side-chain of the backbone. Either side-chainor functional group Y or both may be part of a naturally occurringpolypeptide or may be introduced into a naturally occurring polypeptideor into a polypeptide which, at least partially, does not occurnaturally, prior to the reaction with the functional group X.

Moreover, the polypeptide can be, at least partly, of any human oranimal source. In a preferred embodiment, the polypeptide is of humansource.

The polypeptide may be a cytokine, especially erythropoietin, anantithrombin (AT) such as AT III, an interleukin, especiallyinterleukin-2, IFN-beta, IFN-alpha, G-CSF, CSF, interleukin-6 andtherapeutic antibodies.

According to a preferred embodiment, the polypeptide is an antithrombin(AT), preferably AT III (Levy J H, Weisinger A, Ziomek C A, Echelard Y,Recombinant Antithrombin: Production and Role in CardiovascularDisorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416;Edmunds T, Van Patten S M, Pollock J, Hanson E, Bernasconi R, Higgins E,Manavalan P, Ziomek C, Meade H, McPherson J, Cole E S, TransgenicallyProduced Human Antithrombin: Structural and Functional Comparison toHuman Plasma-Derived Antithrombin, Blood 91, 12 (1998) 4661-4671;Minnema M C, Chang A C K, Jansen P M, Lubbers Y T P, Pratt B M,Whittaker B G, Taylor F B, Hack C E, Friedman B, Recombinant humanantithrombin III improves survival and attenuates inflammatory responsesin baboons lethally challenged with Escherichia coli, Blood 95, 4 (2000)1117-1123; Van Patten S M, Hanson E H, Bernasconi R, Zhang K, ManavalnP, Cole E S, McPherson J M, Edmunds T, Oxidation of Methionine Residuesin Antithrombin, J. Biol. Chemistry 274, 15 (1999) 10268-10276).

According to another preferred embodiment the polypeptide is humanIFN-beta, in particular IFN-beta la (cf. Avonex®, REBIF®) and IFN-beta1b (cf. BETASERON®).

A further preferred polypeptide is human G-CSF (granulocyte colonystimulating factor). See, e.g., Nagata et al., The chromosomal genestructure and two mRNAs for human granulocyte colony-stimulating factor,EMBO J. 5: 575-581, 1986; Souza et al., Recombinant human granulocytecolony-stimulating factor: effects on normal and leukemic myeloid cells,Science 232 (1986) 61-65; and Herman et al., Characterization,formulation, and stability of Neupogen® (Filgrastim), a recombinanthuman granulocyte-colony stimulating factor, in: Formulalion,characterization, and stability of protein drugs, Rodney Pearlman and Y.John Wang, eds., Plenum Press, New York, 1996, 303-328.

If a mixture of at least two different polypeptides is used, the atleast two polypeptides may differ, e.g., in the molecular mass, thenumber and/or sequence of amino acids, different degrees ofglycosilation, the number and/or chemical nature of the substituents orthe number of polypeptide chains linked by suitable chemical-bonds suchas disulfide bridges.

According to a preferred embodiment of the present invention, thereaction product of compound (I) and compound (II), optionally furtherreacted with a crosslinking compound, is isolated, preferably accordingto at least one of the above-mentioned processes, and then reacted witha polypeptide having at least one functional group Y capable of beingreacted with the at least one functional group X of the reaction productof compound (I) and compound (II), optionally further reacted with acrosslinking compound, to form at least one chemical linkage. Functionalgroups Y of polypeptides such as proteins are, e.g.,

or a carbohydrate moiety which may be linked to the polypeptide byN-glycosylation or O-glycosylation.

In the context of the present invention, the term “carbohydrate moiety”refers to hydroxyaldehydes or hydroxyketones as well as to chemicalmodifications thereof (see Römpp Chemielexikon, Thieme Verlag Stuttgart,Germany, 9^(th) edition 1990, Volume 9, pages 2281-2285 and theliterature cited therein). Furthermore, it also refers to derivatives ofnaturally occurring carbohydrate moieties like glucose, galactose,mannose, sialic acid and the like. The term also includes chemicallyoxidized, naturally occurring carbohydrate moieties. The structure ofthe oxidized carbohydrate moiety may be cyclic or linear.

The carbohydrate moiety may be linked directly to the polypeptidebackbone. Preferably, the carbohydrate moiety is part of a carbohydrateside chain. More preferably, the carbohydrate moiety is the terminalmoiety of the carbohydrate side chain.

In an even more preferred embodiment, the carbohydrate moiety is agalactose residue of the carbohydrate side chain, preferably theterminal galactose residue of the carbohydrate side chain. Thisgalactose residue can be made available for reaction with the reactionproduct of compound (I) and compound (II) by removal of terminal sialicacids, followed by oxidation, as described hereinunder.

In a still further preferred embodiment, the reaction product ofcompound (I) and (II) is linked to a sialic acid residue of thecarbohydrate side chains, preferably the terminal sialic acid residue ofthe carbohydrate side chain.

Oxidation of terminal carbohydrate moieties can be performed eitherchemically or enzymatically.

Methods for the chemical oxidation of carbohydrate moieties ofpolypeptides are known in the art and include the treatment withperjodate (Charnow et al., 1992, J. Biol. Chem., 267, 15916-15922).

By chemically oxidizing, it is in principle possible to oxidize anycarbohydrate moiety, being terminally positioned or not. However, bychoosing mild conditions (1 mM periodate, 0° C. in contrast to harshconditions: 10 mM periodate 1 h at room temperature), it is possible topreferably oxidize the terminal sialic acid of a carbohydrate sidechain.

Alternatively, the carbohydrate moiety may be oxidized enzymatically.Enzymes for the oxidation of the individual carbohydrate moieties areknown in the art, e.g. in the case of galactose the enzyme is galactoseoxidase. If it is intended to oxidize terminal galactose moieties, itwill be eventually necessary to remove terminal sialic acids (partiallyor completely) if the polypeptide has been produced in cells capable ofattaching sialic acids to carbohydrate chains, e.g. in mammalian cellsor in cells which have been genetically modified to be capable ofattaching sialic acids to carbohydrate chains. Chemical or enzymaticmethods for the removal of sialic acids are known in the art (Chaplinand Kennedy (eds.), 1996, Carbohydrate Analysis: a practical approach,especially Chapter 5 Montreuill, Glycoproteins, pages 175-177; IRL PressPractical approach series (ISBN 0-947946-44-3)).

Therefore, the present invention also relates to a method as describedabove wherein the reaction product of compound (I) and compound (II) isreacted with the polypeptide via an oxidized carbohydrate moietycomprised in the polypeptide.

According to another preferred embodiment of the present invention, thefunctional group of the polypeptide is the thio group. Therefore, thereaction product of compound (I) and (II) may be linked to thepolypeptide via a thioether group wherein the S atom can be derived fromany thio group comprised in the polypeptide.

The thio group may be present in the polypeptide as such. Moreover, itis possible to introduce a thio group into the polyeptide according to asuitbale method. Among others, chemical methods may be mentioned. If adisulfide bridge is present in the polypeptide, it is possible to reducethe —S—S— structure to get a thio group. It is also possible totransform an amino group present in the polypeptide into a SH group byreaction the polypeptide via the amino group with a compound which hasat least two different functional groups, one of which is capable ofbeing reacted with the amino group and the other is an SH group or aprecursor of an SH group. This modification of an amino group may beregarded as an example where the protein is first reacted with acompound (L) which has at least two different functional groups, one ofwhich is capable of being reacted with the amino group and the other isan SH group, and the resulting reaction product is then reacted with,e.g., a HAS derivative comprising HAS and a compound (D), saidderivative comprising a functional group being capable of reacting withthe SH group. It is also possible to introduce an SH group by mutationof the polypeptide such as by introducing a cystein or a suitable SHfunctional amino acid into the polypeptide or such as removing a cysteinfrom the polypeptide so as to disable another cystein in the polypeptideto form a disulfide bridge.

In the context of this embodiment, it is particularly preferred to reactthe polypeptide with a reaction product which results from the reactionof the reaction product of compounds (I) and (II) with a crosslinkingcompound.

Therefore, the present invention also relates to a method as describedabove wherein the reaction product of compound (I) and compound (II) isreacted with a crosslinking compound and the resulting reaction productis further is reacted with the polypeptide via an oxidized carbohydratemoiety and/or a thio group comprised in the polypeptide.

As an especially preferred polypeptide, erythropoietin (EPO) is used.

Therefore, the present invention also relates to a method as describedabove wherein the polypeptide is erythropoietin.

The EPO can be of any human (see e.g. Inoue, Wada, Takeuchi, 1994, Animproved method for the purification of human erythropoietin with highin vivo activity from the urine of anemic patients, Biol. Pharm. Bull.17(2), 1804; Miyake, Kung, Goldwasser, 1977, Purification of humanerythropoietin, J. Biol. Chem., 252(15), 5558-64) or another mammaliansource and can be obtained by purification from naturally occurringsources like human kidney, embryonic human liver or animal, preferablymonkey kidney. Furthermore, the expression “erythropoietin” or “EPO”encompasses also an EPO variant wherein one or more amino acids (e.g. 1to 25, preferably 1 to 10, more preferred 1 to 5, most preferred 1 or 2)have been exchanged by another amino acid and which exhibitserythropoietic activity (see e.g. EP 640 619 B1). The measurement oferythropoietic activity is described in the art (for measurement ofactivity in vitro see e.g. Fibi et al., 1991, Blood, 77, 1203 ff;Kitamura et al, 1989, J. Cell Phys., 140, 323-334; for measurement ofEPO activity in vivo see Ph. Eur. 2001, 911-917; Ph. Eur. 2000, 1316Erythropoietini solutio concentrata, 780-785; European Pharmacopoeia(1996/2000); European Pharmacopoeia, 1996, Erythropoietin concentratedsolution, Pharmaeuropa, 8, 371-377; Fibi, Hermentin, Pauly, Lauffer,Zettlmeissl., 1995, N- and O-glycosylation muteins of recombinant humanerythropoietin secreted from BHK-21 cells, Blood, 85(5), 1229-36; (EPOand modified EPO forms were injected into female NMRI mice (equalamounts of protein 50 ng/mouse) at day 1, 2 and 3 blood samples weretaken at day 4 and reticulocytes were determined)). Further publicationswhere tests for the measurement of the activity of EPO are describedBarbone, Aparicio, Anderson, Natarajan, Ritchie, 1994, Reticulocytesmeasurements as a bioassay for erythropoietin, J. Pharm. Biomed. Anal.,12(4), 515-22; Bowen, Culligan, Beguin, Kendall, Villis, 1994,Estimation of effective and total erythropoiesis in myelodysplasia usingserum trasferrin receptor and erythropoietin concentrations, withautomated reticulocyte parameters, Leukemi, 8(1), 151-5; Delorme,Lorenzini, Giffin, Martin, Jacobsen, Boone, Elliott, 1992, Role ofglycosylation on the secretion and biological activity oferythropoietin, Biochemistry, 31(41), 9871-6; Higuchi, Oheda, Kuboniwa,Tomonoh, Shimonaka, Ochi, 1992; Role of sugar chains in the expressionof the biological activity of human erythropoietin, J. Biol. Chem.,267(11), 7703-9; Yamaguchi, Akai, Kawanishi, Ueda, Masuda, Sasaki, 1991,effects of site-directed removal of N-glycosylation sites in humanerythropoietin on its production and biological properties, J. Biol.Chem., 266(30), 20434-9; Takeuchi, Inoue, Strickland, Kubota, Wada,Shimizu, Hoshi, Kozutsumi, Takasaki, Kobata, 1989, Relationship betweensugar chain structure and biological activity of recombinant humanerythropoietin produced in Chinese hamster ovary cells, Proc. Natl.Acad. Sci. USA, 85(20), 7819-22; Kurtz, Eckardt, 1989, Assay methods forerythropoietin, Nephron., 51(1), 11-4 (German); Zucali, Sulkowski, 1985,Purification of human urinary erythropoietin on controlled-pore glassand silicic acid, Exp. Hematol., 13(3), 833-7; Krystal, 1983, Physicaland biological characterization of erythroblast enhancing factor (EEF),a late acting erythropoietic stimulator in serum distinct fromerythropoietin, Exp. Hematol., 11(1), 18-31.

Preferably, the EPO is recombinantly produced. This includes theproduction in eukaryotic or prokaryotic cells, preferably mammalian,insect, yeast, bacterial cells or in any other cell type which isconvenient for the recombinant production of EPO. Furthermore, the EPOmay be expressed in transgenic animals (e.g. in body fluids like milk,blood, etc.), in eggs of transgenic birds, especially poultry, preferredchicken, or in transgenic plants.

The recombinant production of a polypeptide is known in the art. Ingeneral, this includes the transfection of host cells with anappropriate expression vector, the cultivation of the host cells underconditions which enable the production of the polypeptide and thepurification of the polypeptide from the host cells. For detainedinformation see e.g. Krystal, Pankratz, Farber, Smart, 1986,Purification of human erythropoietin to homogeneity by a rapid five-stepprocedure, Blood, 67(1), 71-9; Quelle, Caslake, Burkert, Wojchowski,1989, High-level expression and purification f a recombinant humanerythropoietin produced using a baculovirus vector, Blood, 4(2), 652-7;EP 640 619 B1 and EP 668 351 B1.

In a preferred embodiment, the EPO has the amino acid sequence of humanEPO (see EP 148 605 B2).

The EPO may comprise one or more carbohydrate side chains, preferably 1to 12, more preferably 1 to 9, even more preferably 1 to 6 andparticularly 1 to 4, especially preferably 4 carbohydrate side chains,attached to the EPO via N- and/or O-linked glycosylation, i.e. the EPOis glycosylated. Usually, when EPO is produced in eukaryotic cells, thepolypeptide is posttranslationally glycosylated. Consequently, thecarbohydrate side chains may have been attached to the EPO duringbiosynthesis in mammalian, especially human, insect or yeast cells. Thestructure and properties of glycosylated EPO have been extensivelystudied in the art (see EP 428 267 B1; EP 640 619 B1; Rush, Derby,Smith, Merry, Rogers, Rohde, Katta, 1995, Microheterogeneity oferythropoietin carbohydrate structure, Anal Chem., 67(8), 1442-52;Takeuchi, Kobata, 1991, Structures and functional roles of the sugarchains of human erythropoietins, Glycobiology, 1(4), 33746 (Review).

Therefore, the hydroxyalkyl starch derivative according to the presentinvention may comprise at least one, preferably 1 to 12, more preferably1 to 9, even more preferably 1 to 6 and particularly preferably 1 to 4HAS molecules per EPO molecule. The number of HAS-molecules per EPOmolecule can be determined by quantitative carbohydrate compositionalanalysis using GC-MS after hydrolysis of the product and derivatisationof the resulting monosaccharides (see Chaplin and Kennedy (eds.), 1986,Carbohydrate Analysis: a practical approach, IRL Press Practicalapproach series (ISBN 0-947946-44-3), especially Chapter 1,Monosaccharides, page 1-36; Chapter 2, Oligosaccharides, page 37-53,Chapter 3, Neutral Polysaccharides, page 55-96).

According to an especially preferred embodiment of the presentinvention, the carbohydrate moiety linked to EPO, is part of acarbohydrate side chain. More preferably, the carbohydrate moiety is theterminal moiety of the carbohydrate side chain. In an even morepreferred embodiment, the carbohydrate moiety is a galactose residue ofthe carbohydrate side chain, preferably the terminal galactose residueof the carbohydrate side chain. This galactose residue can be madeavailable for reaction with the reaction product of compound (I) andcompound (II) by removal of terminal sialic acids, followed byoxidation, as described hereinunder. In a further preferred embodiment,the reaction product of compound (I) and (II) is linked to a sialic acidresidue of the carbohydrate side chains, preferably the terminal sialicacid residue of the carbohydrate side chain. The sialic acid is oxidizedas described herein.

Particularly preferably this galactose residue is made available forreaction with the reaction product of compounds (I) and (II) or with thereaction product of the reaction of the reaction product of compounds(I) and (II) and a crosslinking compound via functional group X byremoval of terminal sialic acid followed by oxidation.

As mentioned above, the reaction product of compound (I) and compound(II), optionally reacted with a crosslinking compound, may be reactedwith a thio group comprised in EPO.

It is also possible to react the reaction product of compound (I) andcompound (II), optionally reacted with a crosslinking compound, with athio group as well as with a carbohydrate moiety, each of them comprisedin the at least one further compound, preferably a polypeptide, morepreferably erythropoietin.

According to a preferred embodiment, this SH group may be linked to apreferably oxidized carbohydrate moiety, e.g. by using a hydroxylaminederivative, e.g. 2-(aminooxy)ethylmercaptan hydrochloride (Bauer L. etal., 1965, J. Org. Chem., 30, 949) or by using a hydrazide derivative,e.g. thioglycolic acid hydrazide (Whitesides et al., 1977, J. Org.Chem., 42, 332.)

According to a further preferred embodiment, the thio group ispreferably introduced in an oxidized carbohydrate moiety of EPO, morepreferably an oxidized carbohydrate moiety which is part of acarbohydrate side chain of EPO.

Preferably, the thio group is derived from a naturally occurringcysteine or from an added cysteine. More preferably, the EPO has theamino acid sequence of human EPO and the naturally occurring cysteinesare cysteine 29 and/or 33. In a more preferred embodiment, the reactionproduct of compound (I) and compound (II), optionally reacted with acrosslinking compound, is reacted with cysteine 29 whereas cysteine 33is replaced by another amino acid. Alternatively, the reaction productof compound (I) and compound (II), optionally reacted with acrosslinking compound, is reacted with cysteine 33 whereas cysteine 29is replaced by another amino acid.

In the context of the present invention, the term “added cysteines”indicates that the polypeptides, preferably EPO, comprise a cysteineresidue which is not present in the wild-type polypeptide.

In the context of this aspect of the invention, the cysteine may be anadditional amino acid added at the N- or C-terminal end of EPO.

Furthermore, the added cysteine may have been added by replacing anaturally occurring amino acid by cysteine or a suitably substitutedcysteine. Preferably, in the context of this aspect of the invention,the EPO is human EPO and the replaced amino acid residue is serine 126.

The reaction conditions of the reaction of the reaction product ofcompounds (I) and (II), optionally reacted with a crosslinking compound,with the at least one further compound may be adapted to the specificneeds of the respective reaction, such as in the case the at least onefurther compound is a polypeptide or in the case the at least onefurther compound is a crosslinking compound or in the case the at leastone further compound is a reaction product of a crosslinking compoundand a polypeptide. As buffer compounds, at least one of theabove-mentioned compounds may be preferably used. As solvent or mixtureof solvents, at least one of the above-mentioned solvents may bepreferably used. Isolation and/or after-treatment may be carried out,wherein preferred methods are selected from the methods discussed above.

If the reaction product of compound (I) and compound (II) is, forexample, further reacted with a polypeptide as further compound,preferably EPO, water is preferably used as solvent for the reaction.Additionally to water, at least one further solvent may be present. Aspreferred possible further solvent, DMSO, DMF, methanol or ethanol maybe mentioned.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of the reaction product of compound (I) andcompound (II) with a polypeptide, preferably EPO, is carried out in anaqueous system.

As far as the temperatures which are applied during this reaction areconcerned, no specific limitations exist given that the reaction resultsin the desired hydroxyalkyl starch derivative comprising the reactionproduct of compounds (I) and (II) reacted with the polypeptide via theat least one functional group X. The temperature of the reaction ispreferably in the range of from 4 to 37° C., more preferably in therange of from ID to 30° C. especially preferably in the range of from 15to 25° C.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of the reaction product of compound (I) andcompound (II) with the polypeptide is carried out at a temperature offrom 4 to 37° C.

During the course of the reaction the temperature may be varied,preferably in the above-given ranges, or held essentially constant.

The reaction time for reaction of the reaction product of compound (I)and compound (II) with the polypeptide may be adapted to the specificneeds and is generally in the range of from 0.5 to 48 h, preferably inthe range of from 2 to 24 h and especially preferably in the range offrom 10 to 20 h.

The pH value for the reaction of the reaction product of compound (I)and compound (II) with the polypeptide may be adapted to the specificneeds such as the chemical nature of the reactants.

If, e.g., the reaction product of compound (I) and (II) is reacted witha further compound via the reaction of a functional group X which is ahydroxylamino group —O—NH₂ with at least one aldehyd group which iscomprised in the polypeptide, the pH is preferably in the range of from4.5 to 6, more preferably at about 5.5.

If the reaction product of compound (I) and compound (II) is, forexample, further reacted with a crosslinking compound as furthercompound, preferably EPO, water is preferably used as solvent for thereaction. Additionally to water, at least one further solvent may bepresent. As preferred possible further solvent, DMSO, DMF, methanol orethanol may be mentioned.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of the reaction product of compound (I) andcompound (II) with a crosslinking compound is carried out in an aqueoussystem.

As far as the temperatures which are applied during this reaction areconcerned, no specific limitations exist given that the reaction resultsin the desired hydroxyalkyl starch derivative comprising the reactionproduct of compounds (I) and (II) reacted with the crosslinking compoundvia the at least one functional group X. The temperature of the reactionis preferably in the range of from 4 to 37° C., more preferably in therange of from 10 to 30° C. especially preferably in the range of from 15to 25° C.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of the reaction product of compound (I) andcompound (II) with the crosslinking compound is carried out at atemperature of from 4 to 37° C.

During the course of the reaction the temperature may be varied,preferably in the above-given ranges, or held essentially constant.

The reaction time for reaction of the reaction product of compound (I)and compound (II) with the crosslinking compound may be adapted to thespecific needs and is generally in the range of from 10 min to 10 h,preferably of from 20 min to 5 h and more preferably of from 30 min to 2h.

The pH value for the reaction of the reaction product of compound (I)and compound (II) with the crosslinking compound may be adapted to thespecific needs such as the chemical nature of the reactants.

If, e.g., the reaction product of compound (I) and (II) is reacted witha crosslinking compound which is a crosslinking compound via thefunctional group X which is comprised in the reaction product ofcompound (I) and (II) and is an amino group —NH₂, the pH is preferablyin the range of from 7 to 8.5, more preferably at about 7.2.

If the reaction product of the reaction of the reaction product ofcompounds (I) and (II), and a crosslinking compound is, for example,further reacted with a polypeptide, preferably EPO, water is preferablyused as solvent for the reaction. Additionally to water, at least onefurther solvent may be present. As preferred possible further solvent,DMSO, DMF, methanol or ethanol may be mentioned.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of the reaction product of the compound (I)and compound (II) which is further with reacted with a crosslinkingcompound, with a polypeptide is carried out in an aqueous system.

As far as the temperatures which are applied during this reaction areconcerned, no specific limitations exist given that the reaction resultsin the desired hydroxyalkyl starch derivative comprising the reactionproduct of compounds (I) and (II), reacted with a crosslinking compoundand further reacted with a polypeptide via the at least one functionalgroup X comprised in the crosslinking compound. The temperature of thereaction is preferably in the range of from 4 to 37° C., more preferablyin the range of from 10 to 30° C. especially preferably in the range offrom 15 to 25° C.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of the reaction product of compound (I) andcompound (II) which is further reacted with a crosslinking compound,with the polypeptide is carried out at a temperature of from 4 to 37° C.

During the course of the reaction the temperature may be varied,preferably in the above-given ranges, or held essentially constant.

The reaction time for the reaction of the reaction product of compound(I) and compound (II) which is further reacted with a crosslinkingcompound, with the polypeptide may be adapted to the specific needs andis generally in the range of from 0.5 to 48 h, preferably in the rangeof from 2 to 24 h and especially preferably in the range of from 10 to20 h.

The pH value for the reaction of the reaction product of compound (I)and compound (II) which is further reacted with a crosslinking compound,with the polypeptide may be adapted to the specific needs such as thechemical nature of the reactants.

If, e.g., the reaction product of compound (I) and (II) is which isfurther reacted with a crosslinking compound, is reacted with apolypeptide via the functional group X which is comprised in thecrosslinking compound and is an amino group —NH₂, the pH is preferablyin the range of from 7 to 8.5, more preferably at about 7.2.

The suitable pH value of the reaction mixture may be adjusted in eachcase by adding at least one suitable buffer. Among the preferredbuffers, sodium acetate buffer, sodium phosphate buffer, or boratebuffers may be mentioned.

The reaction product resulting from the reaction of the reaction productof compound (I) and compound (II) with the at least one furthercompound, the at least one further compound being either a polypeptideor a crosslinking compound, and the reaction product further comprisingthe compounds resulting from the reactions of compound (I), compound(II), a crosslinking compound and a polypeptide, may be isolated fromthe reaction mixture by at least one suitable method and subjected to atleast one further treatment such as at least an after-treatment such asdialysis and/or lyophilization.

Once the above-mentioned reaction product is formed, it may be isolatedfrom the reaction mixture by at least one suitable method.

Isolation of the reaction product may be carried out by a suitableprocess which may comprise one or more steps.

According to a preferred embodiment of the present invention, where thereaction product does not comprise a polypeptide, the reaction productis first separated off the reaction mixture or the mixture of thereaction mixture by preferably centrifugal filtration. In a second step,the separated reaction product may be subjected to a further treatmentsuch as an after-treatment like dialysis and/or lyophilisation.According to an even more preferred embodiment, the separated reactionproduct is first dialysed, preferably against water, and thenlyophilized until the solvent content of the reaction product issufficiently low according to the desired specifications of the product.

According to another embodiment of the present invention where thereaction product comprises the polypeptide, the reaction product ispreferably isolated as described in Example 7.8.

According to a further embodiment of the present invention, compound(II) is reacted with a further compound prior to the reaction withcompound (I), i.e. a derivate of compound (II) is produced by thereaction of compound (II) via the at least one functional group X withat least one further compound comprising at least one functional groupY, as described above, prior to the reaction with compound (I).

If compound (II) is first reacted with a further compound, preferably apolypeptide, more preferably EPO, water is preferably used as solventfor the reaction. Additionally to water, at least one further solventmay be present. As preferred possible further solvent, DMSO, DMF,methanol and ethanol may be mentioned.

Therefore, the present invention also relates to a method as describedabove wherein the reaction of compound (II), prior to the reaction withcompound (I), with a further compound, preferably a polypeptide, evenmore preferably EPO, is carried out in an aqueous system.

As far as the temperatures which are applied during the reaction areconcerned, no specific limitations exist given that the reaction resultsin the desired derivative of compound (II) comprising the reactionproduct of compound (II) reacted with at least one further compound viathe at least one functional group X, preferably a polypeptide, morepreferably EPO. The temperature of the reaction are preferably in therange of from 4 to 37° C., more preferably in the range of from 10 to30° C. especially preferably in the range of from 15 to 25° C.

Therefore, the present invention also relates to a method as describedabove wherein the reaction compound (II) with the at least one furthercompound is carried out at a temperature of from 4 to 37° C.

During the course of the reaction the temperature may be varied,preferably in the above-given ranges, or held essentially constant.

The reaction time, the pH value for reaction of compound (II) with theat least one further compound may be adapted to the specific needs suchas the chemical nature of the reactants. The suitable pH value of thereaction mixture may be adjusted by adding at least one suitable buffer.Among the preferred buffers, acetate, phosphate, or borate buffers suchas sodium acetate, sodium phosphate, or sodium borate buffers may bementioned.

The reaction product resulting from the reaction of compound (II) withthe at least one further compound may be isolated from the reactionmixture by at least one suitable method and subjected to at least onefurther treatment such as at least an after-treatment such as dialysisand/or lyophilization.

Once the reaction product of reaction compound (II) with the at leastone further compound is formed, it may be isolated from the reactionmixture by at least one suitable method.

Isolation of the reaction product may be carried out by a suitableprocess which may comprise one or more steps as already described above.

If desired and/or necessary, the NH group bridging R′ and R″ of compound(II) may be protected with a suitable protecting group prior to thereaction of compound (II) with the at least one further compound. Asprotecting group, one of the above-mentioned protecting groups may beused. Prior to the reaction of the reaction product of compound (II) andthe at least one further compound such as a polypeptide, preferably EPO,with compound (I), the protecting group is removed by a at least onesuitable method.

If compound (II) is first reacted with a crosslinking compound or areaction product of a crosslinking compound and a polypeptide, allreaction conditions may be adjusted to the specific needs of thesereactions. Among others, the above-mentioned buffer systems and/orsolvents may be used.

In a second step, the reaction product of the reaction of compound (II)with the at least one further compound is reacted with compound (I).

For this reaction, all reaction conditions may be adjusted to thespecific needs of these reactions. Among others, the above-mentionedbuffer systems and/or solvents may be used.

The reaction product resulting from the reaction of the reaction productof compound (II) and the at least one further compound with compound (I)may be isolated from the respective reaction mixture by at least onesuitable method and subjected to at least one further treatment such asat least an after-treatment such as dialysis and/or lyophilization. Inthis context, every suitable method described above may be used.

Generally, isolation of the HAS-polypeptide conjugate, either with orwithout crosslinking compound, can be performed by using knownprocedures for the purification of natural and recombinant polypeptidessuch as size exclusion chromatography, ion-exchange chromatography,RP-HPLC, hydroxyapatite chromatography, hydrophobic interactionchromatography or combinations of at least two methods thereof.

The covalent attachment of HAS to the polypeptide can be verified bycarbohydrate compositional analysis after hydrolysis of the modifiedprotein.

Demonstration of HAS modification at N-linked oligosaccharides of thepolypeptide can be accomplished by removal of the HAS modified N-glycansand observation of the predicted shift to higher mobility in SDS-PAGE+/− Western Blotting analysis.

HAS modification of the polypeptide at cysteine residues can bedemonstrated by the failure to detect the corresponding proteolyticCys-peptide in RP-HPLC and MALDI/TOF-MS in the proteolytic fragments ofthe HAS-modified product (Zhou et al., 1998, Application of capillaryelectrophoresis, liquid chromatography, electrospray-mass spectrometryand matrix-assisted laser desorption/ionization-time of flight-massspectrometry to the characterization of recombinant humanerythropoietin, Electrophoresis, 19(13), 2348-55). The isolation of theHAS-containing fraction after proteolytic digestion of the Cys-modifiedpolypeptide enables the verification in this fraction of thecorresponding peptide by conventional amino acid compositional analysis.

All embodiments disclosed above with respect of the HAS-polypeptide ofthe invention concerning properties of the polypeptide or HAS apply alsoto the method of the invention for the production of a HAS-polypeptideconjugate. Furthermore, all embodiments disclosed above with respect toHAS-EPO or the preparation thereof which relate to peptides in generalor to HAS apply also to the method of the invention for the productionof a HAS-polypeptide conjugate.

According to an especially preferred embodiment of the present inventionhydroxyethyl starch is reacted with a compound (II), preferably selectedfrom the homo- and heterobifunctional compounds described above, and theresulting reaction product is reacted with a glycoprotein, preferablyerythropoietin, preferably with the oxidized terminal carbohydratemoiety of a EPO carbohydrate side chain.

According to another especially preferred embodiment of the presentinvention hydroxyethyl starch is reacted with a compound (II),preferably selected from the homo- and heterobifunctional compoundsdescribed above, to give a first hydroxyethyl starch derivative. Thisfirst hydroxyethyl starch derivative is subsequently reacted with acrosslinking compound to give a second hydroxyethyl starch derivative.This second hydroxyethyl starch derivative is subsequently reacted witha glycoprotein, preferably erythropoietin, preferably with a —SH groupcomprised in the glycoprotein, to give a third hydroxyethyl starchderivative. Preferably, the crosslinking compound is aheterobifunctional compound. More preferably, the crosslinking compoundis reacted with a functional group comprising the structure —NH— whichis comprised in the first hydroxyethyl starch derivative. Morepreferably, this functional group is —NH₂.

One advantage of the present invention is that it is not necessary touse toxicologically critical solvents in at least one reaction step,preferably all reaction steps, the reaction step involved and thus, isnot necessary to remove these solvents after the production process inorder to avoid the contamination of the products with the solventFurthermore, it is not necessary to perform additional quality controlswith respect to residual toxicologically critical solvents. If organicsolvents, preferably in addition to water, are used, it is preferred touse toxicologically uncritical solvents such as ethanol and/orpropylenglycol.

Another advantage of the present invention is that irreversible orreversible structural changes are avoided in the steps where an aqueoussystem is used as solvent which are otherwise induced by organicsolvents. Consequently, polypeptide derivatives obtained according tothe method of the invention are different from those prepared in organicsolvents such as DMSO.

Furthermore, it has been surprisingly observed that the conjugation ofHAS to polypeptides such as EPO in an aqueous solution minimizes oravoids side reactions. Consequently, this embodiment of the method ofthe invention leads to improved hydroxyalkyl starch products with greatpurity.

According to another aspect, the present invention also relates to thehydroxy alkyl starch derivative, obtainable by a process comprisingreacting hydroxyalkyl starch (HAS) of formula (I)

at its reducing end which is not oxidized prior to said reaction, with acompound of formula (II)R′—NH—R″  (II)wherein R₁, R₂ and R₃ are independently hydrogen or a linear or branchedhydroxyalkyl group, and wherein either R′ or R″ or R′ and R″ comprise atleast one functional group X capable of being reacted with at least oneother compound prior to or after the reaction of (I) and (II).

As already described above in the context of the methods of the presentinvention, O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine is used as apreferred compound (II), and hydroxyethyl starch is used as a preferredhydroxyalkyl starch.

Therefore, the present invention also relates to a hydroxyalkyl starchderivative obtainable by a method wherein hydroxyethyl starch is reactedvia its reducing end with O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine.

Depending on the respective reaction conditions, the solvent or solventmixture used and/or the residues R′ and/or R″ it is possible that thehydroxyalkyl starch derivate obtainable by the method or methodsdescribed above may have the following constitutions (IIIa):

Therefore, the present invention also relates to a hydroxyalkyl starchderivative as described above having a constitution according to formula(IIIa).

It is also possible that, e.g. in the case where R′ is hydrogen that thehydroxyalkyl starch derivate obtainable by the method or methodsdescribed above may have the following constitutions (IIIa) or (IIIa)where (IIIa) and (IIIb) may be both present in the reaction mixturehaving a certain equilibrium distribution:

Therefore, the present invention also relates to a hydroxyalkyl starchderivative as described above having a constitution according to formula(IIIb).

Moreover, the present invention also relates to a hydroxyalkyl starchderivative as described above being present in a mixture ofconstitutions according to formulae (IIIa) and (IIIb).

Depending on the reaction conditions and/or the chemical nature ofcompound (II) used for the reaction, the compounds according to formula(IIIa) may be present with the N atom in equatorial or axial positionwhere also a mixture of both forms may be present having a certainequilibrium distribution.

Depending on the reaction conditions and/or the chemical nature ofcompound (II) used for the reaction, the compounds according to formula(IIIb) may be present with the C—N double bond in E or Z conformationwhere also a mixture of both forms may be present having a certainequilibrium distribution.

In some cases it may be desirable to stabilize the compound according toformula (IIIa). This is especially the case where the compound accordingto formula (IIIa) is produced and/or used in an aqueous solution. Asstabilizing method, acylation of the compound according to formula(IIIa) is particularly preferred, especially in the case where R′ ishydrogen. As acylation reagent, all suitable reagents may be used whichresult in the desired hydroxyalkyl starch derivative according toformula (IVa)

According to especially preferred embodiments of the present invention,the residue Ra being part of the acylation reagent is methyl. Asacylation reagents, carboxylic acid anhydrides, carboxylic acid halides,and carboxylic acid active esters are preferably used.

Therefore, the present invention also relates to a hydroxyalkyl starchderivate obtainable by a method as described above wherein saidderivative has a constitution according to formula (IVa).

The acylation is carried at a temperature in the range of from 0 to 30°C., preferably in the range of from 2 to 20° C. and especiallypreferably in the range of from 4 to 10° C.

In other cases it may be desirable to stabilize the compound accordingto formula (IIIb). This is especially the case where the compoundaccording to formula (IIIb) is produced and/or used in an aqueoussolution. As stabilizing method, reduction of the compound according toformula (IIIb) is particularly preferred, especially in the case whereR′ is hydrogen. As reduction reagent, all suitable reagents may be usedwhich result in the desired hydroxyalkyl starch derivative according toformula (IVb)

According to especially preferred embodiments of the present invention,as reduction reagents boro hydrides such as NaCNBH₃ or NaBH₄ are used.

Therefore, the present invention also relates to a hydroxyalkyl starchderivate obtainable by a method as described above wherein saidderivative has a constitution according to formula (IVb).

The reduction is carried at a temperature in the range of from 4 to 100°C., preferably in the range of from 10 to 90° C. and especiallypreferably in the range of from 25 to 80° C.

The present invention further relates to mixtures of compounds (IIIa)and (IIIb), (IVa) and (IVb), (IIIa) and (IVa), (IIIa) and (IVb), (IIIb)and (IVa), (IIIb) and (IVb), (IIIa) and (IIIb) and (IVa), (IIIa) and(IIIb) and (IVb), (IVa) and (IVb) and (IIIa), and (IVa) and (IVb) and(IIIb) wherein (IIIa) and/or (IVa) may be independently present in aconformation where the N atom in equatorial or axial position and/orwherein (IIIb) may be present with the C—N double bond in E or Zconformation.

According to one aspect of the present invention, compound (I) isreacted with compound (II) to give a first reaction product. Said firstreaction product is then optionally stabilized according to at least oneof the methods described above. The first, optionally stabilizedreaction product is then reacted with at least one further compound viathe reaction of at least one functional group X comprised in R″ of thefirst reaction product with at least one functional group Y comprised inthe at least one further compound, to give a second reaction product.Said second reaction product is then optionally stabilized according toat least one of the methods described above.

According to yet another aspect of the present invention, the at leastone further compound is a polypeptide or a crosslinking compound or areaction product of a crosslinking compound with a polypeptide. In casethe at least one further compound is a polypeptide, the functional groupY is comprised in the polypeptide. In case the at least one furthercompound is a crosslinking compound, the functional group Y is comprisedin the crosslinking compound and optionally also in the polypeptide. Incase the at least one further compound is a reaction product of acrosslinking compound with a polypeptide, the functional group Y iscomprised in the crosslinking compound.

According to a further aspect of the present invention, compound (II) isreacted with at least one further compound via the reaction of at leastone functional group X comprised in R″ of compound (II) with at leastone functional group Y comprised in the at least one further compound togive a first reaction product. The at least one further compound ispreferably a polypeptide or a crosslinking compound or a reactionproduct of a crosslinking compound with a polypeptide, as discussedabove. Said first reaction product is then reacted with compound (I) viathe reaction of the reducing end of compound (I) with the NH group ofthe first reaction product bridging the original residues R′ and R″ ofcompound (II) to give a second reaction product. Said second reactionproduct is then optionally stabilized according to at least one of themethods described above.

According to an especially preferred embodiment of the presentinvention, hydroxyethyl starch is used as compound (I),O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine is used as compound (I),and EPO having an oxidized terminal carbohydrate moiety of acarbohydrate side chain is used as further compound. More preferably,hydroxyethyl starch is reacted withO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine to give a firsthydroxyethyl starch derivate, and that first derivative is furtherreacted with EPO having an oxidized terminal carbohydrate moiety of acarbohydrate side chain to give a second hydroxyethyl starch derivate.In this specific case, no stabilizing reaction whatsoever has to becarried out.

Therefore, the present invention also relates to a hydroxyalkyl starchderivative obtainable by a method wherein hydroxyethyl starch is reactedvia its reducing end with O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamineand the reaction product is reacted with erythropietin via the oxidizedterminal carbohydrate moiety of a carbohydrate side chain of theerythropietin.

According to yet another especially preferred embodiment of the presentinvention, hydroxyethyl starch is used as compound (I),O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine is used as compound (II),a heterobifunctional crosslinking compound having a maleimide group anda N-hydroxy succinimide active ester group, is used, and EPO having atleast one —SH group (referred to as ThioEPO) is used as polypeptide.More preferably, hydroxyethyl starch is reacted withO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine to give a firsthydroxyethyl starch derivate, that first derivative is further reactedwith the N-hydroxy succinimide active ester group of the crosslinkingcompound to give a second derivative, and that second derivative isreacted via the maleimide group with the ThioEPO to give a thirdhydroxyethyl starch derivate.

The hydroxyalkyl starch derivative which in the following is referred toas HAS-EPO conjugate and which is formed by reaction of compound (I)with compound (II) and possibly a crosslinking compound anderythrpoietin, has the advantage that it exhibits an improved biologicalstability when compared to the erythropoietin before conjugation. Thisis mainly due to the fact that this hydroxyalkyl starch derivative isless or even not recognized by the removal systems of the liver andkidney and therefore persists in the circulatory system for a longerperiod of time. Furthermore, since the HAS is attachedsite-specifically, the risk of destroying the in-vivo biologicalactivity of EPO by conjugation of HAS to EPO is minimized.

The HAS-EPO conjugate of the invention may exhibit essentially the samein-vitro biological activity as recombinant native EPO, since thein-vitro biological activity only measures binding affinity to the EPOreceptor. Methods for determining the in-vitro biological activity areknown in the art.

Furthermore, the HAS-EPO exhibits a greater in-vivo activity than theEPO used as a starting material for conjugation (unconjugated EPO).Methods for determining the in vivo biological activity are known in theart.

The HAS-EPO conjugate may exhibit an in vivo activity of from 110% to300%, preferably from 110% to 200%, more preferably from 110% to 180% orfrom 110 to 150%, most preferably from 110% to 140%, if the in-vivoactivity of the unconjugated EPO is set as 100%.

Compared to the highly sialylated EPO of Amgen (see EP 428 267 B1), theHAS-EPO exhibits preferably at least 50%, more preferably at least 70%,even more preferably at least 85% or at least 95%, at least 150%, atleast 200% or at least 300% of the in vivo activity of the highlysialylated EPO if the in-vivo activity of highly sialylated EPO is setas 100%. Most preferably, it exhibits at least 95% of the in vivoactivity of the highly sialylated EPO.

The high in-vivo biological activity of the HAS-EPO conjugate of theinvention mainly results from the fact that the HAS-EPO conjugateremains longer in the circulation than the unconjugated EPO because itis less recognized by the removal systems of the liver and because renalclearance is reduced due to the higher molecular weight. Methods for thedetermination of the in-vivo half life time of EPO in the circulationare known in the art (Sytkowski, Lunn, Davis, Feldman, Siekman, 1998,Human erythropoietin dimers with markedly enhanced in vivo activity,Proc. Natl. Acad. Sci. USA, 95(3), 11848).

Consequently, it is a great advantage of the present invention that aHAS-EPO conjugate is provided which may be administered less frequentlythan the EPO preparations commercially available at present Whilestandard EPO preparations have to be administered at least every 3 days,the HAS-EPO conjugate of the invention is preferable administered twicea week, more preferably once a week.

Furthermore, the method of the invention has the advantage that aneffective EPO derivative can be produced at reduced costs since themethod does not comprise extensive and time consuming purification stepsresulting in low final yield, e.g. it is not necessary to purify awayunder-sialylated EPO forms which are known to exhibit low or no in-vivobiological activity.

Furthermore, the present invention relates to a pharmaceuticalcomposition comprising, in a therapeutically effective amount, theHAS-polypeptide conjugate, preferably the HAS-EPO conjugate, morepreferably the HES-EPO conjugate of the present invention. In apreferred embodiment, the pharmaceutical composition comprises furtherat least one pharmaceutically acceptable diluent, adjuvant and/orcarrier useful in erythropoietin therapy.

Therefore, the present invention also relates to a pharmaceuticalcomposition comprising, in a therapeutically effective amount, ahydroxyalkyl starch derivative as described above wherein the reactionproduct of compound (I) with compound (II) is reacted via the at leastone functional group X comprised in compound (II) with at least onefurther compound or wherein compound (I) is reacted via the at least onefunctional group X with at least one further compound prior to thereaction with compound (I) and wherein the at least one further compoundis a polypeptide.

According to preferred embodiments of the present invention, thepolypeptide, preferably erythropoietin is reacted with compound (II) orwith the reaction product of compound (I) and compound (II) via a thiogroup or an oxidized carbohydrate moiety comprised in the polypeptide.

According to an even more preferred embodiment of the present invention,the polypeptide, preferably erythropoietin is reacted with compound (II)or with the reaction product of compound (I) and compound (II) via anoxidized carbohydrate moiety comprised in the polypeptide.

Therefore, the present invention relates to a pharmaceutical compositionas described above wherein the polypeptide is reacted with compound (II)or with the reaction product of compound (I) and compound (II) via anoxidized carbohydrate moiety comprised in the polypeptide.

According to preferred embodiments, the polypeptide is GCS-F, AT III,IFN-beta or erythropoietin, more preferably erythropoietin.

Therefore, the present invention also relates to a pharmaceuticalcomposition as described above wherein the polypeptide iserythropoietin.

According to an especially preferred embodiment of the presentinvention, the pharmaceutical composition as described above is producedby reacting hydroxyethyl starch in an aqueous medium with a compoundaccording to the following formula

and by reacting the reaction product with erythropoietin.

According to a particularly preferred embodiment, the erythropoietin isoxidised with sodium periodate prior to the aformentioned reaction.

According to another particularly preferred embodiment, theerythropoietin is partially desialylated and subsequently oxidised withsodium periodate prior to the reaction.

According to a further preferred embodiment of the present invention,pharmaceutical compositions comprising a hydroxyalkyl starch derivativewhich are produced on the basis of a completely reduced Thio-EPOaccording to Example 5 are excluded.

According to another preferred embodiment, the present invention alsorelates to a pharmaceutical composition comprising, in a therapeuticallyeffective amount, a hydroxyalkyl starch derivative as described abovewherein the reaction product of compound (I) with compound (II) isreacted via the at least one functional group X comprised in compound(II) with at least one further compound or wherein compound (II) isreacted via the at least one functional group X with at least onefurther compound prior to the reaction with compound (I) and wherein theat least one further compound is a crosslinking compound and thereaction product of the reaction product of compounds (I) and (II) withthe crosslinking compound is reacted with a polypeptide.

According to a still further preferred embodiment, the present inventionrelates to the aforementioned pharmaceutical composition wherein thepolypeptide is erythropoietin.

The above-mentioned pharmaceutical composition is especially suitablefor the treatment of anemic disorders or hematopoietic dysfunctiondisorders or diseases related thereto.

A “therapeutically effective amount” as used herein refers to thatamount which provides therapeutic effect for a given condition andadministration regimen. The administration of erythropoietin isoforms ispreferably by parenteral routes. The specific route chosen will dependupon the condition being treated. The administration of erythropoietinisoforms is preferably done as part of a formulation containing asuitable carrier, such as human serum albumin, a suitable diluent, suchas a buffered saline solution, and/or a suitable adjuvant. The requireddosage will be in amounts sufficient to raise the hematocrit of patientsand will vary depending upon the severity of the condition beingtreated, the method of administration used and the like.

The object of the treatment with the pharmaceutical composition of theinvention is preferably an increase of the hemoglobin value of more than6.8 mmol/I in the blood. For this, the pharmaceutical composition may beadministered in a way that the hemoglobin value increases between from0.6 mmol/l and 1.6 mmol/l per weeks If the hemoglobin value exceeds 8.7mmol/l, the therapy should be preferably interrupted until thehemoglobin value is below 8.1 mmol/l.

The composition of the invention is preferably used in a formulationsuitable for subcutaneous or intravenous or parenteral injection. Forthis, suitable excipients and carriers are e.g. sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium chlorate, polysorbate 80,HSA and water for injection. The composition may be administered threetimes a week, preferably two times a week, more preferably once a week,and most preferably every two weeks.

Preferably, the pharmaceutical composition is administered in an amountof 0.01-10 μg/kg body weight of the patient, more preferably 0.1 to 5μg/kg, 0.1 to 1 μg/kg, or 0.2-0.9 μg/kg, most preferably 0.3-0.7 μg/kg,and most preferred 0.4-0.6 μg/kg body weight.

In general, preferably between 10 μg and 200 μg, preferably between 15μg and 100 μg are administered per dosis.

The invention further relates to a HAS-polypeptide according to thepresent invention for use in method for treatment of the human or animalbody.

The invention further relates to the use of a HAS-EPO conjugate of thepresent invention for the preparation of a medicament for the treatmentof anemic disorders or hematopoietic dysfunction disorders or diseasesrelated hereto.

The invention is further illustrated by the following examples, tables,and figures which are in no way intended to restrict the scope of thepresent invention.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1

FIG. 1 shows an SDS page analysis of the HES-EPO conjugate, producedaccording to example 4.1.

-   Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co,    Karlsruhe, D); molecular weights (in kD) of the protein marker from    top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.-   Lane B: Crude product after conjugation according to example 4.1.-   Lane C: EPO starting material.

FIG. 2

FIG. 2 shows an SDS page analysis of the HES-EPO conjugate, producedaccording to example 4.3.

-   Lane A: Crude product after conjugation according to example 4.3.-   Lane B: EPO starting material.-   Lane C: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co,    Karlsruhe, D); molecular weights (in kD) of the protein marker from    top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.

FIG. 3

FIG. 3 shows an SDS page analysis of HES-EPO conjugates, producedaccording to examples 6.1 and 6.4.

-   Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co,    Karlsruhe, D); molecular weights (in kD) of the protein marker from    top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.-   Lane B: Crude product after conjugation according to example 6.4.-   Lane C: Crude product after conjugation according to example 6.1.-   Lane D: EPO starting material.

FIG. 4

FIG. 4 shows an SDS page analysis of HES-EPO conjugates, producedaccording to examples 6.2, 6.3, 6.5, and 6.6.

-   Lane A: Protein marker Roti®-Mark PRESTAINED (Carl Roth GmbH+Co,    Karlsruhe, D); molecular weights (in kD) of the protein marker from    top to bottom: 245, 123, 77, 42, 30, 25.4, and 17.-   Lane B: Crude product after conjugation according to example 6.6,    based on Example 1.3 b).-   Lane C: Crude product after conjugation according to example 6.5,    based on Example 1.1 b).-   Lane D: Crude product after conjugation according to example 6.6,    based on Example 1.3 a).-   Lane E: Crude product after conjugation according to example 6.5,    based on Example 1.1 a).-   Lane F: Crude product after conjugation according to example 6.2.-   Lane G: Crude product after conjugation according to example 6.3.-   Lane K: EPO starting material.

FIG. 5

SDS-PAGE analyses of EPO-GT-1 subjected to mild acid treatment for 5min.=lane 2; 10 min.=lane 3; 60 min.=lane 4 and untreated EPO=lane 1;the mobility shift of EPO after removal of N-glycans is shown (+PNGASE).

FIG. 6

HPAEC-PAD pattern of oligosaccharides isolated from untreated EPO andfrom EPO incubated for 5 min., 10 min. and 60 min. under mild acidhydrolysis conditions. Roman numbers I-V indicate the elution positionof I=desialylated diantennary structure, II=trisialylated triantennarystructures (two isomers), III=tetrasialylated tetraantennary structure+2N-acetyllactosamine repeats, IV=tetrasialylated tetraantennarystructure+1 N-acetyllactosamine repeat; V=tetrasialylated tetraantennarystructure+without N-acetyllactosamine repeat. The elution area ofoligosaccharides structures without, with 1-4 sialic acid is indicatedby brackets.

FIG. 7

HPAEC-PAD of N-linked oligosaccharides after desialylation; the elutionposition of N-acetylneuraminic acid is shown; numbers 1-9 indicate theelution position of standard oligosaccharides: 1=diantennary;2=triantennary (24 isomer), 3=triantennary (2-6 isomer);4=tetraantennary; 5=triantennary plus 1 repeat; 6=tetraantennary plus 1repeat; 7=triantennary plus 2 repeats; 8=tetraantennary plus 2 repeatsand 9=tetraantennary plus 3 repeats.

FIG. 8

SDS-PAGE analysis of mild treated and untreated EPO which were subjectedto periodate oxidation of sialic acid residues. 1=periodate oxidizedwithout acid treatment; 2=periodate oxidized 5 min. acid treatment;3=periodate oxidized and acid treatment 10 min.; 4=periodate oxidizedwithout acid treatment; 5=BRP EPO standard without periodate and withoutacid treatment.

FIG. 9

HPAEC-PAD pattern of native oligosaccharides isolated from untreated EPOand from EPO incubated for 5 min and 10 min under mild acid hydrolysisconditions and subsequent periodate treatment. The elution area ofoligosaccharides structures without and with 1-4 sialic acid isindicated by brackets 1-5.

FIG. 10

SDS-PAGE analysis of the time course of HES-modification of EPO-GT-1-A:20 μg aliquots of EPO-GT-1-A were reacted with hydroxylamine-modifiedHES derivative X for 30 min, 2, 4 and 17 hours. Lane 1=30 min reactiontime; land 2=2 hour reaction time; land 3=4 hours reaction time; lane4=17 hours reaction time; lane 5=EPO-GT-1-A without HES-modification.Left figure shows the shift in mobility of EPO-GT-1-A with increasingincubation time in the presence of the with hydroxylamine-modified HESderivative (flow rate: 1 ml·min⁻¹) X: Lane 1=30 min reaction time; lane2=2 hours reaction time; lane 3=4 hours reaction time, land 4=17 hoursreaction time; lane 5=EPO-GT-1-A with HES modification. The figure onthe right shows analysis of the same samples after their treatment withN-glycosidase.

FIG. 11

SDS-PAGE analysis of Q-Sepharose fractions of HES-EPO conjugates. Each1% of the flow-through and 1% of the fraction eluting at high saltconcentrations were concentrated in a Speed Vac concentrator and wereloaded onto the gels in sample buffer. EPO protein was stained byCoomassie Blue. A=sample I; B=sample II; C=sample III; K=controlEPO-GT-1; A1, B1, C1 and K1 indicated the flow-through fraction; A2, B2,C2 and K2 indicates the fraction eluted with high salt concentration.

FIG. 12 a

SDS-PAGE analysis of HES-modified EPO sample A2 (see FIG. 7), controlEPO sample K2 and EPO-GT-1-A EPO preparation were digested in thepresence of N-glycosidase in order to remove N-linked oligosaccharides.All EPO samples showed the mobility shift towards low molecular weightforms lacking or containing O-glycan. A lower ratio of theO-glycosylated and nonglycosylated protein band was observed for theHES-modified EPO sample A2 after de-N-glycosylation and a diffuseprotein band was detected around 30 KDa, presumably representingHES-modification at the sialic acid of O-glycan residue (see arrowmarked by an asterisk).

FIG. 12 b

SDS-PAGE analysis after mild hydrolysis of HES-modified EPO sample A2(see FIG. 11), control EPO sample K2 and EPO-GT-1A which were untreatedor digested in the presence of N-glycosidase in order to remove N-linkedoligosaccharides (see FIG. 12 a). Both high molecular weight form of A2before and A after N.glycosidase treatment (see brackets with andwithout arrow) disappeared upon acid treatment of the samples. The BRPEPO standard which was run for comparison was not subjected to mild acidtreatment.

FIG. 13

HPAEC-PAD analysis of N-linked oligosaccharide material liberated fromHES-modified sample A, from EPO-GT-1-A and from a control EPO sampleincubated with unmodified HES (K). Roman numbers I-V indicate theelution position of I=disialylated diantennary structure,II=trisialylated triantennary structures (two isomers),III=tetrasialylated tetraantennary structure+2 N-acetyllactosaminerepeats, IV=tetrasialylated tetraantennary structure+1N-acetyllactosamine repeat, V=tetrasialylated tetraantennarystructure+without N-acetyllactosamine repeat; brackets indicate theelution area of di-, tri- and tetrasialylated N-glycans as reported inthe legends of FIGS. 6 and 9.

FIG. 14

HPAEC-PAD analysis of N-linked oligosaccharide material liberated fromHES-modified sample A, from EPO-OT-1A and from a control EPO sample (K)incubated with unmodified HES. The retention times of a mixture ofstandard oligosaccharides is shown: numbers 1-9 indicate the elutionposition of standard oligosaccharides: 1=diantennary; 2=triantennary(2-4 isomer); 3=triantennary (2-6 isomer); 4=tetraantennary;5=triantennary plus 1 repeat, 6=tetraantennary plus 1 repeat;7=triantennary plus 2 repeats; 8=tetraantennary plus 2 repeats and9=tetraantennary plus 3 repeats.

FIGS. 15 to 21

FIGS. 15 to 21 represent MALDI/TOF mass spectra of the enzymaticallyliberated and chemically desialylated N-glycans isolated fromHES-modified EPO and control EPO preparations. Major signals at m/z1809.7, 2174.8, 2539.9, 2905.0 and 3270.1 ([M+Na]⁺) correspond to di- totetraantennary complex-type N-glycan structures with no, one or twoN-acetyllactosamine repeats accompanied by weak signals due to loss offucose or galactose which are due to acid hydrolysis conditions employedfor the desialylation of samples for MS analysis.

FIG. 15

MALDI/TOF spectrum: desialylated oligosaccharides of HES-modified EPOA2.

FIG. 16

MALDI/TOF spectrum: desialylated oligosaccharides of EPO GT-1-A.

FIG. 17

MALDI/TOF spectrum: desialylated oligosaccharides of EPO K2.

FIG. 18

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1.

FIG. 19

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjectedto acid hydrolysis for 5 min.

FIG. 20

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjectedto acid hydrolysis for 10 min.

FIG. 21

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 subjectedto acid hydrolysis for 60 min.

In the context of the present invention, the degree of substitution,denoted as DS, relates to the molar substitution, as described above(see also Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278,in particular p. 273). Throughout the invention, the DS of the HES18/04when measured according to Sommermeyer et al., 1987,Krankenhauspharmazie, 8(8), 271-278 was 0.5.

EXAMPLES Example 1 Formation of Hydroxyethyl Starch Derivatives byReductive Amination Example 1.1 Reaction of Hydroxyethyl Starch with1,3-diamino-2-hydroxy propane

-   a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (MW=18,000    D, DS=0.4)) in 5 ml water, 0.83 mmol 1,3-diamino-2-hydroxy propane    and 50 mg sodium cyanoborohydrate NaCNBH₃ were added. The resulting    mixture was incubated at 80° C. for 17 h. The reaction mixture was    added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v).    The precipitate was collected by centrifugation and dialysed for 4 d    against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio    Science Deutschland GmbH, Bonn, D), and lyophilized.-   b) Incubation of the mixture resulting from adding 0.83 mmol    1,3-diamino-2-hydroxy propane and 50 mg sodium cyanoborohydrate    NaCNBH₃ to the solution of 200 mg hydroxyethyl starch was also    possible and carried out at 25° C. for 3 d.

Example 1.2 Reaction of Hydroxyethyl Starch with 1,2-dihydroxy-3-aminopropane

-   a) To a solution of 200 mg hydroxyethyl starch (HES18/0:4 (MW 18,000    D, DS=0.4)) in 5 ml water, 0.83 mmol. 1,2-dihydroxy-3-amino propane    and 50 mg sodium cyanoborohydrate NaCNBH₃ were added. The resulting    mixture was incubated at 80° C. for 17 h. The reaction mixture was    added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v).    The precipitate was collected by centrifugation and dialysed for 4 d    against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio    Science Deutschland GmbH, Bonn, D), and lyophilized.-   b) Incubation of the mixture resulting from adding 0.83 mmol    1,2-dihydroxy-3-amino propane and 50 mg sodium cyanoborohydrate    NaCNBH₃ to the solution of 200 mg hydroxyethyl starch was also    possible and carried out at 25° C. for 3 d.

The reaction of 1,2-dihydroxy-3-amino propane with HES was confirmedindirectly by quantification of formaldehyde, resulting from theoxidative cleavage of the 1,2-diole in the reaction product by periodateas described by G. Avigad, Anal. Biochem. 134 (1983) 449-504.

Example 1.3 Reaction of Hydroxyethyl Starch with 1,4-diamino butane

-   a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (MW=18,000    D, DS=0.4)) in 5 ml water, 0.83 mmol 1,4-diamino butane and 50 mg    sodium cyanoborohydrate NaCNBH₃ were added. The resulting mixture    was incubated at 80° C. for 17 h. The reaction mixture was added to    160 ml of a cold 1:1 mixture of acetone and ethanol (v/v). The    precipitate was collected by centrifugation and dialysed for 4 d    against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio    Science Deutschland GmbH, Bonn, D), and lyophilized.-   b) Incubation of the mixture resulting from adding 0.83 mmol    1,4-diamino butane and 50 mg sodium cyanoborohydrate NaCNBH₃ to the    solution of 200 mg hydroxyethyl starch was also possible and carried    out at 25° C. for 3 d.

Example 1.4 Reaction of Hydroxyethyl Starch with 1-mercapto-2-aminoethane

-   a) To a solution of 200 mg hydroxyethyl starch (HES18/0.4 (MW 18,000    D, DS=0.4)) in 5 ml water, 0.83 mmol 1-mercapto-2-amino ethane and    50 mg sodium cyanoborohydrate NaCNBH₃ were added. The resulting    mixture was incubated at 80° C. for 17 h. The reaction mixture was    added to 160 ml of a cold 1:1 mixture of acetone and ethanol (v/v).    The precipitate was collected by centrifugation and dialysed for 4 d    against water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio    Science Deutschland GmbH, Bonn, D), and lyophilized.)-   b) Incubation of the mixture resulting from adding 0.83 mmol    1-mercapto-2-amino ethane and 50 mg sodium cyanoborohydrate NaCNBH₃    to the solution of 200 mg hydroxyethyl starch was also possible and    carried out at 25° C. for 3 d.

Example 2 Formation of Hydroxyethyl Starch Derivatives by ConjugationExample 2.1 Reaction of Hydroxyethyl Starch with Carbohydrazide

0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in 8 ml aqueous0.1 M sodium acetate buffer, pH 5.2, and 8 mmol carbohydrazide (SigmaAldrich, Taufkirchen, D) were added. After stirring for 18 h at 25° C.,the reaction mixture was added to 160 ml of a cold 1:1 mixture ofacetone and ethanol (v/v). The precipitated product was collected bycentrifugation, re-dissolved in 40 ml water, and dialysed for 3 dagainst water (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio ScienceDeutschland GmbH, Bonn, D), and lyophilized.

Example 2.2 Reaction of Hydroxyethyl Starch with Adepic Dihydrazide

0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in 8 ml aqueous0.1 M sodium acetate buffer, pH 5.2, and 8 mmol adepic dihydrazide(Lancaster Synthesis, Frankfurt/Main, D) were added. After stirring for18 h at 25° C., the reaction mixture was added to 160 ml of a cold 1:1mixture of acetone and ethanol (v/v). The precipitated product wascollected by centrifugation, re-dissolved in 40 ml water, and dialysedfor 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, PerbioScience Deutschland GmbH, Bonn, D), and lyophilized.

Example 2.3 Reaction of Hydroxyethyl Starch with1,4-phenylene-bis-3-thiosemicarbazide

0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in 8 mmolaqueous 0.1 M sodium acetate buffer, pH 5.2, and 8 mmol1,4-phenylene-bis-3-thiosemicarbazide (Lancaster Synthesis,Frankfurt/Main, D) were added. After stirring for 18 h at 25° C., 8 mlwater was added to the reaction mixture, and the suspension wascentrifugated for 15 min at 4,500 rpm. The clear supernatant wasdecanted and subsequently added to 160 ml of a cold 1:1 mixture ofacetone and ethanol (v/v). The precipitated product was collected bycentrifugation, re-dissolved in 40 ml water, and centrifugated for 15min at 4,500 rpm. The clear supernatant was dialysed for 3 d againstwater (SnakeSkin dialysis tubing, 3.5 KD cut off, Perbio ScienceDeutschland GmbH, Bonn, D), and lyophilized.

Example 2.4 Reaction of Hydroxyethyl Starch withO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine

O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine was synthesized asdescribed in Boturyn et al. Tetrahedron 53 (1997) p. 5485-5492 in 2steps from commercially available materials.

0.96 g of HES18/0.4 (MW=18,000 D, DS=0.4) were dissolved in 8 ml aqueous0.1 M sodium acetate buffer, pH 5.2, and 8 mmolO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine were added. After stirringfor 18 h at 25° C., the reaction mixture was added to 160 ml of a cold1:1 mixture of acetone and ethanol (v/v). The precipitated product wascollected by centrifugation, re-dissolved in 40 ml water, and dialysedfor 3 d against water (SnakeSkin dialysis tubing, 3.5 KD cut off, PerbioScience Deutschland GmbH, Bonn, D), and lyophilized.

Example 3 Oxidation of Erythropoietin

Oxidized erythropoietin was produced as described in Example 7. Asoxidised erythropoietin, EPO-GT-1-A as described in Example 7.11(c) wasused (EPO-GT-1 without acid hydrolysis, treated with mild periodateoxidation).

Example 4 Conjugation of Hydroxyethyl Starch Derivatives with OxidizedErythropoietin of Example 3 Example 4.1 Reaction of OxidizedErythropoietin with the Reaction Product of Example 2.1

Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18μl of a solution of the HES derivate as produced according to example2.1 (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) wasadded, and the mixture was incubated for 16 h at 25° C. Afterlyophilisation, the crude product was analyzed by SDS-Page with NuPAGE10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif., USA) asdescribed in the instructions given by Invitrogen. The gel is stainedwith Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D)overnight.

The experimental result is shown in FIG. 1. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 4.2 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 23

Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18μl of a solution of the HES derivate as produced according to example2.3 (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) wasadded, and the mixture was incubated for 16 h at 25° C. Afterlyophilisation, the crude product was analyzed by SDS-Page with NuPAGE10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif., USA) asdescribed in the instructions given by Invitrogen

Example 4.3 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 2.4

Oxidized EPO (1.055 μg/μl) in 20 mM PBS buffer was adjusted to pH 5.3with 5 M sodium acetate buffer, pH 5.2. To 19 μl of the EPO solution, 18μl of a solution of the HES derivate as produced according to example2.4 (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer, pH 5.2) wasadded, and the mixture was incubated for 16 h at 25° C. Afterlyophilisation, the crude product was analyzed by SDS-Page with NuPAGE10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, Calif., USA) asdescribed in the instructions given by Invitrogen. The gel is stainedwith Roti-Blue Coomassie staining reagent (Roth, Karlsruhe, D) overnight

The experimental result is shown in FIG. 2. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 5 Formation of Thio-EPO by Reduction of Erythropoietin

241.5 μg erythropoietin (EPO-GT-1, see Example 7) in 500 μl of a 0.1 Msodium borate buffer, 5 mM EDTA, 10 mM DTT (Lancaster, Morcambe, UK), pH8.3, were incubated for 1 h at 37° C. The DTT was removed by centrifugalfiltration with a VIVASPIN 0.5 ml concentrator, 10 KD MWCO (VIVASCIENCE,Hannover, D) at 13,000 rpm, subsequent washing 3 times with the boratebuffer and twice with a phosphate buffer (0.1 M, 9.15 MNaCl, 50 mM EDTA,pH 7.2).

Example 6 Conjugation of Hydroxyethyl Starch Derivatives withThio-Erythropoietin Using a Crosslinking Compound

In each of the following examples, N-(alpha-maleimidoacetoxy)succinimide ester (AMAS)

was used as crosslinking compound.

Example 6.1 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 2.1 and the Crosslinking Compound

To 50 nmol HES derivate as produced according to example 2.1 anddissolved in 200 μl of a 0.1 M sodium phosphate buffer (6.1 M, 9.15 MNaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (SigmaAldrich, Taufkirchen, D) in DMSO were added. The clear solution wasincubated for 80 min at 25° C. and 20 min at 40° C. Remaining AMAS wasremoved by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30min each with the phosphate buffer.

To the residual solution, 15 μg of ThioEPO as produced according toexample 5 (1 μg/μl in phosphate buffer) were added, and the mixture wasincubated for 16 h at 25° C. After lyophilisation, the crude product wasanalysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer(Invitrogen, Carlsbad, USA) as described in the instructions given byInvitrogen The gel is stained with Roti-Blue Coomassie staining reagent(Roth, Karlsruhe, D) overnight.

The experimental result is shown in FIG. 3. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 6.2 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 2.2 and the Crosslinking Compound

To 50 nmol HES derivate as produced according to example 2.2 anddissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 MNaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (SigmaAldrich, Taufkirchen, D) in DMSO were added. The clear solution wasincubated for 80 min at 25° C. and 20 min at 40° C. Remaining AMAS wasremoved by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30min each with the phosphate buffer.

To the residual solution, 15 μg of ThioEPO as produced according toexample 5 (1 μg/μl in phosphate buffer) were added, and the mixture wasincubated for 16 h at 25° C. After lyophilisation, the crude product wasanalysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer(Invitrogen, Carlsbad, USA) as described in the instructions given byInvitrogen. The gel is stained with Roti-Blue Coomassie staining reagent(Roth, Karlsruhe, D) overnight.

The experimental result is shown in FIG. 4. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 6.3 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 2.3 and the Crosslinking Compound

To 50 nmol HES derivate as produced according to example 2.3 anddissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 MNaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (SigmaAldrich, Taufkirchen, D) in DMSO were added. The clear solution wasincubated for 80 min at 25° C. and 20 min at 40° C. Remaining AMAS wasremoved by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30min each with the phosphate buffer.

To the residual solution, 15 μg of ThioEPO as produced according toexample 5 (1 μg/μl in phosphate buffer) were added, and the mixture wasincubated for 16 h at 25° C. After lyophilisation, the crude product wasanalysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer(Invitrogen, Carlsbad, USA) as described in the instructions given byInvitrogen. The gel is stained with Roti-Blue Coomassie staining reagent(Roth, Karlsruhe, D) overnight.

The experimental result is shown in FIG. 4. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 6.4 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 2.4 and the Crosslinking Compound

To 50 nmol HES derivate as produced according to example 2.4 anddissolved in 200 μl of a 0.1 M sodium phosphate buffer (0.1 M, 9.15 MNaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 μmol AMAS (SigmaAldrich, Taufkirchen, D) in DMSO were added. The clear solution wasincubated for 80 min at 25° C. and 20 min at 40° C. Remaining AMAS wasremoved by centrifugal filtration with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D) at 13,000 rpm, washing 4 times and 30min each with the phosphate buffer.

To the residual solution, 15 μg of ThioEPO as produced according toexample 5 (1 μg/μl in phosphate buffer) were added, and the mixture wasincubated for 16 h at 25° C. After lyophilisation, the crude product wasanalysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer(Invitrogen, Carlsbad, USA) as described in the instructions given byInvitrogen. The gel is stained with Roti-Blue Coomassie staining reagent(Roth, Karlsruhe, D) overnight.

The experimental result is shown in FIG. 3. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 6.5 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 1.1 and the Crosslinking Compound

To 50 nmol HES derivate as produced according to example 1.1, atincubation conditions of 80° C. and 17 h (Example 1.1 a)) as well as of25° C. and 3 d (Example 1.1 b)), and dissolved in 200 μl of a 0.1 Msodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μlof a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSOwere added. The clear solution was incubated for 80 min at 25° C. and 20min at 40° C. Remaining AMAS was removed by centrifugal filtration witha VIVASPIN 0.5 ml concentrator, 5 KD MWCO VIVASCIENCE, Hannover, D) at13,000 rpm, washing 4 times and 30 min each with the phosphate buffer.

To the residual solution, 15 μg of ThioEPO as produced according toexample 5 (1 μg/μl in phosphate buffer) were added, and the mixture wasincubated for 16 h at 25° C. After lyophilisation, the crude product wasanalysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer(Invitrogen, Carlsbad, USA) as described in the instructions given byInvitrogen. The gel is stained with Roti-Blue Coomassie staining reagent(Roth, Karlsruhe, D) overnight.

The experimental result is shown in FIG. 4. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 6.6 Reaction of Oxidized Erythropoietin with the ReactionProduct of Example 1.3 and the Crosslinking Compound

To 50 nmol HES derivate as produced according to example 1.3, atincubation conditions of 80° C. and 17 h (Example 1.3 a)) as well as of25° C. and 3 d (Example 1.3 b), and dissolved in 200 μl of a 0.1 Msodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 μM EDTA, pH 7.2), 10 μlof a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSOwere added. The clear solution was incubated for 80 min at 25° C. and 20min at 40 IC. Remaining AMAS was removed by centrifugal filtration witha VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D) at13,000 rpm, washing 4 times and 30 min each with the phosphate buffer.

To the residual solution, 15 μg of ThioEPO as produced according toexample 5 (1 μg/μl in phosphate buffer) were added, and the mixture wasincubated for 16 h at 25° C. After lyophilisation, the crude product wasanalysed by SDS-Page with NuPAGE 10% Bis-Tris Gels/MOPS buffer(Invitrogen, Carlsbad, USA) as described in the instructions given byInvitrogen. The gel is stained with Roti-Blue Coomassie staining reagent(Roth, Karlsruhe, D) overnight.

The experimental result is shown in FIG. 4. A successful conjugation isindicated by the migration of the protein band to higher molecularweights. The increased bandwidth is due to the molecular weightdistribution of the HES derivatives used and the number of HESderivatives linked to the protein.

Example 7 Preparative Production of HES-EPO Conjugates

Summary

HES-EPO conjugates were synthesized by coupling of HES derivatives(average mw of 18,000 Dalton; hydroxyethyl substitution degree of 0.4)to the partially (mild periodate) oxidized sialic acid residues on theoligosaccharide chains of recombinant human EPO. Based on carbohydratestructural analysis the modifications introduced did not affect thestructural integrity of the core oligosaccharide chains sinceMALDI/TOF-MS of the mild acid treated HES-modified glycans revealedintact neutral N-acetyllactosamine-type chains which wereindistinguishable from those observed in unmodified EPO product. Theresults obtained indicate that at least 3 modified HES-residues areattached per EPO molecule in the case of the EPO preparation which wassubjected to modification without prior partial sialic acid removal. AnEPO variant lacking about 50% of the sialic acid residues of the formerprotein showed a similar apparent high molecular weight mobility inSDS-PAGE (60-110 KDa vs 40 KDa for the BRP EPO standard). The HESmodified EPO is stable under standard ion-exchange chromatographyconditions at room temperature at pH 3-10.

The EPO-bioassay in the normocythaemic mouse system indicates that theHES-modified EPO has 2.5-3.0 fold higher specific activity (IU/mg) inthis assay when compared to the International BRP EPO reference standardbased on protein determination using the UV absorption value from theEuropean Pharmacopeia and an RP-HPLC EPO protein determination methodcalibrated against the BRP EPO standard preparation

Example 7.1 Materials and Methods

(a) Liberation of N-Linked Oligosaccharides by Digestion withN-Glycosidase

Samples were incubated with 25 units (according to manufacturer'sspecification, Roche Diagnostics, Germany) of recombinant PNGase F overnight at 37° C. Complete digestion was monitored by the specificmobility shift of the protein in SDS-PAGE. The released N-glycans wereseparated from the polypeptide by addition of 3 volumes of cold 100%ethanol and incubation at −20° C. for at least 2 hours (Schroeter S etal., 1999). The precipitated protein was removed by centrifugation for10 minutes at 4° C. at 13000 rpm. The pellet was then subjected to twoadditional washes with 500 μl of ice-cold 75% ethanol. Theoligosaccharides in the pooled supernatants were dried in a vacuumcentrifuge (Speed Vac concentrator, Savant Instruments Inc., USA). Theglycan samples were desalted using Hypercarb cartridges (25 mg or 100 mgof HyperCarb) as follows prior to use: the columns were washed with3×500 μl of 80% acetonitrile (v/v) in 0.1% TFA followed by washes with3×500 μl of water. The samples were diluted with water to a final volumeof 300 μl-600 μl before loading onto the cartridge which then wasrigorously washed with water. Oligosaccharides were eluted with 1.2 ml(25 mg cartridges; 1.8 ml in the case of 100 mg cartridges) 25%acetonitrile in water containing 0.1% trifluoroacetic acid (v/v). Theeluted oligosaccharides were neutralized with 2 M NH₄OH and were driedin a Speed Vac concentrator. In some cases desalting of N-glycosidasereleased oligosaccharides was performed by adsorption of the digestionmixture from samples <100 μg of total (glyco)protein onto 100 mgHypercarb cartridges.

(b) Analysis of Oligosaccharides by Matrix-Assisted LaserDesorption/Ionization Time-of-Flight Mass-Spectrometry(MALDI/TOF/TOF-MS)

A Bruker ULITRAFLEX time-of-flight (TOF/TOF) instrument was used: nativedesialylated oligosaccharides were analyzed using 2,5-dihydroxybenzoicacid as UV-absorbing material in the positive as well as in the negativeion mode using the reflectron in both cases. For MS-MS analyses,selected parent ions were subjected to laser induced dissociation (LID)and the resulting fragment ions separated by the second TOF stage (LIFT)of the instrument Sample solutions of 1 μl and an approximateconcentration of 1-10 pmol·μl⁻¹ were mixed with equal amounts of therespective matrix. This mixture was spotted onto a stainless steeltarget and dried at room temperature before analysis.

Example 7.2 Preparation and Characterization of Recombinant Human EPO(EPO-GT-1)

EPO was expressed from recombinant CHO cells as described (Mueller P Pet al., 1999, Dorner A J et al., 1984) and the preparations werecharacterized according to methods described in the Eur. Phar. (Ph. Eur.4, Monography January 2002:1316: Erythropoietin concentrated solution).The final product had a sialic acid content of 12 nMol (+/− 1.5 nMol)per nMol of protein. The structures of N-linked oligosaccharides weredetermined by HPAEC-PAD and by MALDI/TOF-MS as described. (Nimtz et al.,1999, Grabenhorst, 1999). The EPO preparations that were obtainedcontained di-, tri- and tetrasialylated oligosaccharides (2-12%, 15-28%and 60-80%, respectively, sulphated and pentasialylated chains werepresent in small amounts). The overall glycosylation characteristics ofEPO preparations were similar to that of the international BRP EPOstandard preparation.

The isoelectric focusing pattern of the recombinant EPO was comparableto that of the international BRP Reference EPO standard preparationshowing the corresponding isoforms. 25% of the EPO protein lackedO-glycosylation at Ser₁₂₆ of the polypeptide chain.

Example 7.3 Preparation of Partially Desialylated EPO Forms

EPO GT-1 protein (2.84 mg/ml) was heated to 80° C. in 20 mM Na-phosphatebuffer pH 7.0 and then 100 μl of 1 N H₂SO₄ was added per 1 ml of the EPOsolution; incubation was continued for 5 min, 10 min and 60 min,respectively, yielding EPO preparations of different degree ofsialylation. Quantitation of oligosaccharides with 0-4 sialic acids wasperformed after liberation of oligosaccharides with polypeptideN-glycosidase and isolation of N-linked chains was performed bydesalting using Hypercarb cartridges (25 mg HyperSep Hypercarb;ThermoHypersil-Keystone, UK). EPO preparations were neutralized byaddition of 1 N NaOH and were frozen in liquid N₂ and were stored at−20° C. until further use.

Example 7.4 Periodate Oxidation of Sialylated EPO Forms

To 10 mg of untreated or mild acid treated EPO dissolved in 3.5 ml of 20mM Na-phosphate buffer pH 7.0 was added 1.5 ml of 0.1 M Na-acetatebuffer pH 5.5 and the mixture was cooled to 0° C. in an ice-bath; 500 μlof 10 mM Na-periodate was added and the reaction mixture was kept in thedark for 60 min at 0° C. Then 10 μl of glycerol was added and incubationwas continued for further 10 min in the dark. The partially oxidized EPOforms were separated from reagents by desalting using VIVASPINconcentrators (10,000 MWCO, PES Vivascience AG, Hannover, Germany)according to manufacturer's recommendation at 3000 rpm in a laboratorycentrifuge equipped with a fixed angle rotor. After freezing in liquidnitrogen the EPO preparations were stored in a final volume of 4 ml at−20° C.

100 μg aliquots of the partially oxidized EPO preparation were subjectedto N-glycosidase treatment and oligosaccharides were isolated usingHypercarb cartridges as described. Oligosaccharides were desialylated bymild acid treatment and were analyzed by HPAEC-PAD and their retentiontimes were compared to those of authentic standard oligosaccharides asdescribed (Nimtz et al., 1990 and 1993).

Example 7.5 Reduction of EPO Disulfides with Dithioerythreitol

5 mg of EPO-GT-1 was incubated in 5 ml of 0.1 M Tris/HCl buffer pH 8.1in the presence of 30 mM dithioerythreitol (DTT) at 37° C. for 60minutes; removal of DTT was achieved by using a Vivaspin concentrator at4° C., 4 cycles of buffer exchange. The final reduced EPO preparationwas frozen in liquid nitrogen and stored at −20° C. in 50 mM Na-acetatebuffer pH 5.5.

Example 7.6 EPO Protein Determination

Quantitative determination of EPO protein was performed by measuring UVabsorption at 280 nm according to the Eur. Phar. (European Pharmacopeia4, Monography January 2002: 1316: erythropoietin concentrated solution)in a cuvette with 1 cm path length. In addition, EPO was quantitated byapplying a RP-HPLC method using a RP-C4 column (Vydac Protein C4, Cat.#214TP5410, Grace Vydac, Ca, US); the HPLC method was calibrated usingthe erythropoietin BRP 1 reference standard (European Pharmacopeia,Conseil de l'Europe B.P. 907-F67029, Strasbourg Cedex 1).

Example 7.7 Oxidation of Desialylated EPO with Galactose Oxidase

4.485 mg of completely desialylated EPO was incubated in 20 mMNa-phosphate buffer pH 6.8 in the presence of 16 μl catalase (6214units/200 ml) and 80 μl of galactose oxidase (2250 units/ml fromDactyliuni dendiroides (Sigma-Aldrich, Steinheim, Germany); incubationat 37° C. was over night; 2 times 20 μl of galactose oxidase was addedafter 4 hours and after 8 hours after starting of the incubation.

Example 7.8 Preparation of EPO Samples for Bioassays

Purification of EPO Front Incubations of Periodate- orGalactose-Oxidase-Oxidized EPO Protein Preparations with Activated HES

Purification of EPO samples (removal of unreacted HES derivatives) wascarried out at room temperature. The EPO incubation mixtures(approximately 5 mg of EPO protein) were diluted 1:10 with buffer A (20mM N-morpholine propane sulfonic acid [MOPS/NaOH] in H₂O bidest, pH 8.0)and were applied to a column containing 3 ml Q-Sepharose HP (PharmaciaCode no. 17-1014-03, Lot no. 220211) equilibrated with 10 column volumes(CV) of buffer A by using a flow rate of 0.5 ml/min. The column waswashed with 6-8 CV of buffer A (flow rate=0.8 ml/min) and elution wasperformed by using buffer B (20 mM morpholine ethane sulfonic acid[MES/NaOH], 0.5 M NaCl in H₂O bidest, pH 6.5) at a flow rate of 0.5ml/min. EPO was detected by UV absorption at 280 nm and eluted in about6 ml. The column was regenerated by using 3 CV of buffer C (20 mM MES,1.5 M NaCl in H₂O adjusted to pH 6.5) and was re-equilibrated by using10 CV of buffer A (flow rate 0.7 ml/min).

Buffer exchange of EPO eluates obtained from the Q-Sepharose step wasperformed using Vivaspin concentrators and phosphate buffered saline(PBS) with each 3 centrifugation cycles per sample; samples wereadjusted to 2 ml with PBS and were stored at −20° C.

Only <25% of the partially desialylated and subsequently mild periodateoxidized EPO forms that were subjected to HES-modification were obtainedfrom the Q-Sepharose eluate since under the conditions employed thebasic EPO forms did not bind Q-Sepharose and were found in theflow-through together with nonreacted HES derivatives.

Example 7.9 High-pH Anion-Exchange Chromatography with PulsedAmperometric Detection (HPAEC-PAD)

Purified native and desialylated oligosaccharides were analyzed byhigh-pH anion-exchange (HPAE) chromatography using a Dionex BioLC system(Dionex, USA) equipped with a CarboPac PA1 column (0.4×25 cm) incombination with a pulsed amperometric detector (PAD) (Schröter et al.,1999; Nimtz et al., 1999). Detector potentials (E) and pulse durations Mwere: E1: +50 mV, T1: 480 ms; E2: +500 mV, T2: 120 ms; E3: −500 mV, T3:60 ms, and the output range was 500-1500 nA. The oligosaccharides werethen injected onto the CarboPac PA1 column which was equilibrated with100% solvent A. For desialylated oligosaccharides elution (flow rate: 1ml·min⁻¹) was performed by applying a linear gradient (0-20%) of solventB over a period of 40 min followed by a linear increase from 20-100%solvent B over 5 min. Solvent A was 0.2 M NaOH in bidistilled H₂O,solvent B consisted of 0.6 M NaOAc in solvent A. For nativeoligosaccharides the column was equilibrated with 100% solvent C (0.1 MNaOH in bidistilled H₂O) and elution (flow rate: 1 ml·min⁻¹) wasperformed by applying a linear gradient (0-35%) of solvent D over aperiod of 48 min followed by a linear increase from 35-100% solvent Dover 10 min. Solvent D consisted of 0.6 M NaAc in solvent C.

Example 7.10 Monosaccharide Compositional Analysis of N-Glycans,HES-Modified N-glycans and EPO Protein by GC-MS

Monosaccharides were analyzed as the corresponding methyl glycosidesafter methanolysis, N-reacetylation and trimethylsilylation by GC/MS[Chaplin, M. F. (1982) A rapid and sensitive method for the analysis ofcarbohydrate. Anal. Biochem. 123, 336-341]. The analyses were performedon a Finnigan GCQ ion trap mass spectrometer (Finnigan MAT corp., SanJose, Calif.) running in the positive ion-E1-mode equipped with a 30 mDB5 capillary column. Temperature program: 2 min isotherm at 80° C.,then 10 degrees min⁻¹ to 300° C.

Monosaccharides were identified by their retention time andcharacteristic fragmentation pattern. The uncorrected results ofelectronic peak integration were used for quantification.Monosaccharides yielding more than one peak due to anomericity and/orthe presence of furanoid and pyranoid forms were quantified by addingall major peaks. 0.5 μg of myo-inositol was used as an internal standardcompound.

Example 7.11 Results Example 7.11(a) Characterization of N-Glycans ofMild Acid Treated (Partially Desialylated) EPO-GT-1

EPO-GT-1 preparations subjected to mild acid treatment for 5, 10 or 60mL were analyzed by SDS-PAGE before and after liberation of N-linkedoligosaccharides by incubation with N-glycosidase as shown in FIG. 5.N-linked oligosaccharides were subjected to HPAEC-PAD oligosaccharidemapping (FIG. 6). The untreated EPO-GT-1 contained >90% of N-linkedoligosaccharides with 3 or 4 sialic acid residues whereas after 5 min.of incubation in the presence of mild acid <40% of carbohydrate chainshad 3 or 4 sialic acid residues. HPAEC-PAD of the desialylated N-glycansrevealed that the ratio of neutral oligosaccharides that were detectedfor the untreated EPO-GT-1 and remained stable in the preparationssubjected to acid treatment for 5, 10 or 60 min. MALDI/TOF-MS of thedesialylated glycans revealed that <90% of the proximal fucose waspresent after mild acid treatment of the protein.

Example 7.11(b) Characterization of Periodate Treated EPO-GT-1

SDS-PAGE mobility of mild periodate treated EPO forms that werepreviously subjected to a 5 and 10 minute treatment with acid or werenot treated are compared in FIG. 8. The conditions used for periodateoxidation of sialic acids did not change the SDS-PAGE pattern of EPOpreparations (compare FIG. 5). Oxidation of sialic acids resulted in ashift of oligosaccharides in HPAEC-PAD analysis to earlier elution times(compare FIGS. 6 and 9).

Example 7.11(c) Characterization of HES-Modified EPO Derivatives

(aa) Time Course of HES Modification of EPO-GT-1-A withHydroxylamine-Modified HES Derivative X, Produced According to Example2.4

400 μg of hydroxylamine-modified HES derivative X was added to 20 μg ofEPO-GT-1-A (mild periodate oxidized EPO, not acid hydrolyzed prior tomild periodate oxidation) in 20 μL of 0.5 M NaOAc buffer pH 5.5 and thereaction was stopped after 30 min, 2, 4, and 17 hours, respectively, byfreezing samples in liquid nitrogen. Subsequently samples were stored at−20° C. until further analysis.

SDS-PAGE sample buffer was added and the samples were heated to 90° C.and applied onto SDS-gels. As shown in FIG. 10, increasing incubationtimes resulted in an increased shift towards higher molecular weight ofthe protein. After 17 hours of incubation in the presence of thehydroxylamine-modified HES derivative X a diffuse Coomassie stainedprotein band was detected migrating in an area between 60 and 11 KDa,based on the position of molecular weight standards (see left part ofFIG. 10). Upon treatment with N-glycosidase most of the protein wasshifted towards the position of de-N-glycosylated EPO (see FIG. 10,right gel; arrow A indicates migration position of N-glycosidase, arrowB indicates migration position of de-N-glycosylated EPO; the diffuseprotein band visible in the region between the 28 KDa and 36 KDamolecular weight standards presumably represents EPO-forms which aremodified by HES and the O-glycosylation site of the molecule. In view ofthe specificity of N-glycosidase we conclude from this result that infact HES-modification occurs at the periodate oxidized sialic acidresidues of glycans of the EPO protein.

(bb) Characterization of HES-EPO Conjugates

HES-EPO conjugates I (originating from EPO-GT-1 after mild periodateoxidation, i.e. from EPO-GT-1-A), II (resulting from EPO-GT-1 subjectedto 5 min acid hydrolysis and Mild periodate oxidation), m (resultingfrom EPO-GT-1 subjected to 10 min acid hydrolysis and mild periodateoxidation) were synthesized as described before. A control incubation(K) was included containing unmodified EPO-GT-1 under the same bufferconditions to which an equivalent amount of unmodified HES was added.The incubation mixtures were subjected to further purification forsubsequent biochemical analysis of the HES-EPO derivatives.

Incubations HES-EPO conjugates I, II and III as well as the controlincubation K were subjected to a Q-Sepharose purification step asdescribed under “Material and Methods” (Example 7.8) in order to removethe excess of nonreacted HES-reagent which was expected in flow throughof the ion-exchange column. Due to the high amounts of basic EPO formscontained in previously acid treated samples II and III we expectedconsiderable amounts of modified EPO product from these incubations inthe flow through. As is shown in FIG. 11, almost all of the EPO materialfrom samples I was retained by Q-Sepharose column whereas onlyapproximately 20-30% of the samples III and II was recovered in thefraction eluting with high salt concentration. All of the proteinmaterial from the incubations with HES derivative X, both in theflow-through and the fractions eluting with high salt, had apparenthigher molecular weight in SDS-PAGE when compared to the control EPO.

In order to characterize in more detail the HES-modified EPO sample Aand K (see FIG. 11) were compared to periodate oxidized form EPO-GT-1-A.The samples were subjected to N-glycosidase treatment and as is depictedin FIGS. 12 a and 12 b the release of N-glycans resulted in the two lowmolecular weight bands at the position of the O-glycosylated andnonglycosylated EPO forms of the standard EPO preparation. In the caseof sample A a further band migrating at the position of the 28 KDa mwstandard was detected suggesting HES-modification at the O-glycan ofthis EPO variant (cf. Example 7.11(c)(aa)). This band (and also theheavily HES-modified high mw form of N-glycosylated EPO, see FIGS. 12 aand 12 b) disappeared after subjecting the samples to mild hydrolysiswhich is in agreement with the view that HES modification was achievedat the periodate oxidised sialic acid residues of erythropoietin.

Aliquots of the N-glycosidase incubation mixtures were hydrolyzed usingconditions enabling the complete removal of sialic acids residues (andalso the sialic acid linked HES derivative) from oligosaccharides; afterneutralization, the mixtures were then absorbed onto small Hypercarbcolumns for their desalting. The columns were washed rigorously withwater followed by elution of bound neutral oligosaccharides with 40%acetonitile in H₂O containing 0.1% of trifuloacetic acid. The resultingoligosaccharides were subjected to MALDI/TOF-MS. The spectra of thedesialylated oligosaccharide fractions from sample A, EPO-GT-1-A andsample K showed identical masses for complex type oligosaccharides atm/z=1810 Da (diantennary), 2175=triantennary, 2540=tetraantennary,2906=tetraantennary plus 1 N-acetyllactosamine repeat and3271=tetraantennary plus 2 N-acetyllactosamine repeats; small signalscorresponding to lack of fucose (−146) and galactose (minus 162) weredetected which are attributable to the acid hydrolysis conditionsapplied for sialic acid removal (see MALDI—Figures 15, 16 and 17).

In a parallel experiment the N-glycosidase digestion mixture wasabsorbed onto 1 ml RP-C18 cartridge (without prior acid hydrolysis ofoligosaccharides) and elution was performed with 5% acetonitrile inwater containing 0.1% TFA; under these conditions the EPO protein wascompletely retained onto the RP-material and oligosaccharides werewashed off from the column with 5% acetonitrile in H₂O containing 0.1%TFA. The de-N-glycosylated EPO protein was eluted with 70% acetonitrilein H₂O containing 0.1% TFA. The oligosaccharide fractions from theRP-C18 step of N-glycosidase-treated sample A, EPO GT-1-A and sample Kwere neutralized and subjected to desalting using Hypercarb cartridgesas described before. The isolated oligosaccharides were subjected toHPAEC-PAD mapping before (see FIG. 13) and after mild acid treatmentunder conditions which enabled quantitative removal of sialic acids fromglycans (see FIG. 14).

The HPAEC-PAD profile for the native material obtained from theHES-modified sample A showed only neglectable signals foroligosaccharides whereas EPO GT-1-A-derived oligosaccharides exhibitedthe same glycan profile as the one shown in FIG. 9 (sample namedEPO-GT-1 after mild periodate treatment). The elution profile ofoligosaccharides obtained from the control EPO sample (K) yielded theexpected pattern (compare profile in FIG. 6). For comparison, the nativeoligosaccharide profile of the international BRP-EPO standard isincluded for comparison and as reference standard.

After mild acid hydrolysis, all oligosaccharide preparations showed anidentical elution profile of neutral oligosaccharide structures (seeFIG. 14) with the expected qualitative and quantitative compositon ofdi-, tri- and tetraantennary complex-type carbohydrate chains asdescribed in the methods section for the EPO preparation which was usedas a starting material in the present study. This result demonstratesthat the HES-modification of the EPO sample results in a covalentlinkage of the HES derivative which is detached from the EPO-protein byN-glycosidase and is acid-labile since it is removed from the N-glycansusing mild acid treatment conditions known to desialylate carbohydrates(see FIGS. 12 a+b).

(cc) Monosaccharide Compositional Analysis of HES-EPO and HES-EPON-Glycans by GC-MS

In order to further confirm HES-modification of EPO at the N-glycans ofthe molecule, EPO samples were digested with N-glycosidase and the EPOprotein was adsorbed onto RP-C18 cartridges whereas oligosaccharidematerial was washed off as described above. As shown in Table 2, glucoseand hydroxyethylated glucose derivatives were detected only in the EPOprotein which was subjected to HES-modification at cysteine residues andin oligosaccharide fractions of EPO sample A2.

Example 7.11(d) In-vivo Assay of the Biological Activity of HES-ModifiedEPO

The EPO-bioassay in the normocythaemic mouse system indicates wasperformed according to the procedures described in the EuropeanPharmacopeia; the laboratory that carried out the EPO assay was usingthe International BRP EPO reference standard preparation. For theHES-modified EPO A2 preparation a mean value for the specific activityof 294,600 units per mg EPO of protein was determined indicating anapproximately 3-fold higher specific activity when compared to theInternational BRP EPO reference standard preparation that was includedin the samples sent for activity assays.

The results of the study are summarized in Table 3.

REFERENCES

-   Nimtz M, Noll G, Paques E P, Conradt H S.-   Carbohydrate structures of a human tissue plasminogen activator    expressed in recombinant Chinese hamster ovary cells.-   FEBS Lett 1990 Oct. 1; 271(1-2):14-8-   Dorner A J, Wasley L C, Kaufman R J.-   Increased synthesis of secreted proteins induces expression of    glucose-regulated proteins in butyrate-treated Chinese hamster ovary    cells.-   J Biol Chem. 1989 Dec. 5; 264 (34):20602-7-   Mueller P P, Schlenke P, Nintz M, Conradt H S, Hauser H-   Recombinant glycoprotein quality in proliferation-controlled BHK-21    cells.-   Biotechnol Bioeng. 1999 Dec. 5; 65(5):529-36-   Nimtz M, Martin W, Wray V, Kloppel K D, Augustin J, Conradt H S.-   Structures of sialylated oligosaccharides of human erythropoietin    expressed in recobminant BHK-21 cells.-   Eur J Biochem. 1993 Apr. 1; 213(1)-39-56-   Hermentin P, Witzel R, Vliegenthart J F, Kamerling J P, Nimtz M,    Conradt H S.-   A strategy for the mapping of N-glycans by high-ph anion-exchange    chromatography with pulsed amperometric detection.-   Anal Biochem. 1992 June; 203(2):281-9-   Schroter S, Derr P, Conradt H S, Nimtz M, Hale G, Kirchhoff C.-   Male specific modification of human CD52.-   J Biol Chem. 1999 Oct. 15; 274(42):29862-73

TABLE 1 Abre- via- tion Chemical Name Type AMAS N-(α-Maleimidoacetoxy)succinimide ester E

BMPH N-(β- Maleimidopropionic acid) hydrazide TFA A

BMPS N-(β- Maleimidopropyloxy) succininaide ester E

EMCH N-(ε-Maleimidocaproic acid) hydrazide A

BMCS N-(ε- Maleimidocaproyloxy) succinimide ester E

GMBS N-γ- Maleimidobutyryloxy- succinimide ester E

KMUH N-(κ- Maleimidoundecanoic acid) hydrazide A

LC- SMCC Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy-(6-amido-Caproate) E

LC- SPDP Succinimidyl 6-(3′-[2- pyridyl- dithio]propionamido) hexanoateF

MBS m-Maleimidobenzoyl-N- hydroxysuccinimide ester E

M₂C₂H 4-(N-Maleimidomethyl)- cyclohexane-1-carboxyl-hydrazide.HCP.½dioxane A

MPBH 4-(4-N- Maleimidophenyl)- butyric acid hydazide.HCl A

SATA N-Succinimidyl S-acetylthio-acetate H

SATP N-Succinimidyl S-acetylthio-propionate H

SBAP Succinimidyl 3- (bromoacetamido) propionate D

SIA N-Succinimidyl iodoacetate C

SIAB N-Succinimidyl (4-iodoacetyl) aminobenzoate C

SMCC Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate E

SMPB Succinimidyl 4-(p- maleimidophenyl) butyrate E

SMPH Succinimidyl-6-(β -maleimidopropion- amido) hexanoate E

SMPT 4-Succinimidyloxy- carbonyl-methyl-α-(2- pyridyldithio)toluene F

SPDP N-Succinimidyl 3-(2- pyridyldithio)propionate F

Sulfo- EMCS N-(ε- Maleimidocaproyloxy) sulfosuccinimide ester E

Sulfo- GMBS N-γ- Maleimidobutryloxy- sulfosuccinimide ester E

Sulfo- KMUS N-(κ-Maleimidoundecan oyloxy)sulfosuccinimide ester E

Sulfo- LC- SPDP Sulfosuccinimidyl6-(3′-[2-pyridyl- dithio]propionamido)hexanoate F

Sulfo- MBS m-Maleimidobenzoyl-N- hydroxysulfosuccinimide ester E

Sulfo- SIAB Sulfosuccinimidyl(4- iodoacetyl) aminobenzoate C

Sulfo- SMCC Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate E

Sulfo- SMPB Sulfosuccinimidyl 4-(p-maleimidophenyl) butylate B

Sulfo- LC-SMPT Suflosuccinimidyl 6-(α-methyl-α-[2- pyridyldithio]-toluamido)hexanoate F

SVSB N-Succinimidyl-(4- vinylsulfonyl)benzoate G

TABLE 2 Monosaccharide compositional analysis of glycans fromHES-modified EPO and control samples I. II. III. III. IV. V. VI. GlycansGlycans Glycans Glycans Glycans Glycans Cystein **Mono- from from fromfrom from EPO- from modified saccharide A2 EPO-GT-1A K2 A2 GT-1A K2 EPOprotein* fucose 1,935 3,924 2,602 2,246 4,461 2,601 2,181 mannose 6,02811,020 9,198 6,379 11,668 6,117 6,260 galactose 8,886 19,935 14,42710,570 16,911 11,555 10,386 glucose 17,968 — — 21,193 trace trace 33,021GlcNAc 7,839 21,310 14,440 11,360 15,953 10,503 10,498 GlcHe1 5,583 — —5,926 — — 14,857 GlcHe2 1,380 — — 1,552 — — 3,775 NeuNAc 5,461 822 4,5043,895 4,871 13,562 13,003 inositol 1,230 2,310 1,620 2,050 1,320 1,1341,087 *the equivalent of Cys-HES-modified EPO protein was subjected tocompositional analysis; the EPO protein was isolated from theHES-incubation mixture by chromatography on a Q-Sepharose column asdescribed above and was desalted by centrifugation using a Vivaspin 5separation device. **Monosaccharide determinations were performed fromsingle GC runs of the pertrimethylsilylated methylglycosides; theelectronical integration values of peaks are given without correctionfor losses during the derivatisation procedure and recoveries of eachcompound.

TABLE 3 Calculated specific activity of EPO sample Sample (based on A280nm and No. Sample description RP-HPLC determination) 850247 1.HES-modified EPO A2 344,000 U/mg 850248 2. EPO-GT-1-A 82,268 U/mg 8502493. Control EPO K2 121,410 U/mg 850250 4. BRP EPO standard 86,702 U/mg850251 1. diluted with 4 volume of PBS 309,129 U/mg 850252 2. dilutedwith 4 volume of PBS 94,500 U/mg 850253 3. diluted with 4 volume of PBS114,100 U/mg 850254 4. diluted with 4 volume of PBS 81,200 U/mg850255 1. diluted with 4 volume of PBS 230,720 U/mg

1. A hydroxyethylstarch (HES) derivative, obtainable by a methodcomprising selectively reacting HES of formula (I)

at its reducing end which is not oxidized prior to said reaction, withO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine, wherein R₁, R₂ and R₃are independently hydrogen or a 2-hydroxyethyl group, and wherein one—O—NH₂ group of the O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine,having the structure

is reacted with compound (I) at its reducing end which is not oxidized.2. A HES derivative as claimed in claim 1, wherein said derivative has aconstitution according to formula


3. A HES derivative as claimed in claim 1, wherein the derivative iseither a compound of formula

or a compound of formula

or a mixture thereof.
 4. A HES derivative wherein the compound asclaimed in claim 1 is reacted via its unreacted O—NH₂ group with atleast one further compound, or wherein theO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine is first reacted at oneof its O—NH₂ groups with at least one further compound and then reactedat its remaining O—NH₂ group with compound (I), wherein the at least onefurther compound is selected from the group consisting of a polypeptideand a crosslinking compound.
 5. A HES derivative as claimed in claim 4wherein the at least one further compound is reacted with one O—NH₂group of O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine or with theunreacted O—NH₂ group of the compound of claim 1 via a thio group or anoxidized carbohydrate moiety comprised in the at least one furthercompound.
 6. A HES derivative as claimed in claim 5 wherein the at leastone further compound is a polypeptide.
 7. A HES derivative as claimed inclaim 6 wherein the polypeptide is erythropoietin.
 8. A HES derivativeas claimed in claim 4 wherein the at least one further compound is acrosslinking compound.
 9. A HES derivative wherein the compound asclaimed in claim 8 is reacted via the crosslinking compound with asecond further compound which is a polypeptide.
 10. A HES derivative asclaimed in claim 9 wherein the second further compound is reacted via athio group or an oxidized carbohydrate moiety comprised in the secondfurther compound.
 11. A HES derivative as claimed in claim 4 wherein thecrosslinking compound is first conjugated to a polypeptide.
 12. Apharmaceutical composition comprising, in a therapeutically effectiveamount, a HES derivative as claimed in claim 4 wherein the at least onefurther compound is a polypeptide.
 13. A pharmaceutical composition asclaimed in claim 12 wherein the polypeptide is reacted with one O—NH₂group of the O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine, or with theunreacted O—NH₂ group of the reaction product of claim 1, via anoxidized carbohydrate moiety comprised in the polypeptide.
 14. Apharmaceutical composition as claimed in claim 13 wherein thepolypeptide is erythropoietin.
 15. A pharmaceutical composition asclaimed in claim 14 wherein HES is reacted in an aqueous medium with theO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine, and the reaction productis reacted with erythropoietin.
 16. A pharmaceutical composition asclaimed in claim 15 wherein the erythropoietin is oxidized with sodiumperiodate prior to the reaction.
 17. A pharmaceutical composition asclaimed in claim 15 wherein the erythropoietin is partially desialylatedand subsequently oxidized with sodium periodate prior to the reaction.18. A pharmaceutical composition comprising, in a therapeuticallyeffective amount, a compound resulting from the reaction of a HESderivative as claimed in claim 4 further reacted with a polypeptide,wherein the at least one further compound is a crosslinking compound.19. A pharmaceutical composition as claimed in claim 18 wherein thepolypeptide is erythropoietin.
 20. A HES derivative obtainable by amethod comprising selectively reacting HES of formula (I)

at its reducing end which is not oxidized prior to said reaction, in anaqueous medium with O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine,wherein R₁, R₂ and R₃ are independently hydrogen or a 2-hydroxyethylgroup, and wherein one O—NH₂ group of theO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine is reacted with compound(I) at its reducing end which is not oxidized.
 21. A HES derivative,wherein the compound as claimed in claim 20 is reacted in an aqueousmedium at its unreacted O—NH₂ group with a polypeptide via a thio groupor an oxidized carbohydrate moiety comprised in the polypeptide.
 22. AHES derivative as claimed in claim 21 wherein the polypeptide iserythropoietin.
 23. A HES derivative wherein one O—NH₂ group ofO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine is reacted in an aqueousmedium with a polypeptide via a thio group or an oxidized carbohydratemoiety comprised in the polypeptide, and the resulting reaction productis then reacted via the unreacted O—NH₂ group comprised in theO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine with HES of formula (I)


24. A HES derivative as claimed in claim 23 wherein the polypeptide iserythropoietin.
 25. A HES derivative obtainable by a method comprisingselectively reacting HES of formula (I)

at its reducing end which is not oxidized prior to said reaction, in anaqueous medium with O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl aminewherein R₁, R₂ and R₃ are independently hydrogen or a 2-hydroxyethylgroup, and reacting the reaction product via its unreacted O—NH₂ groupin an aqueous medium with erythropoietin via an oxidized carbohydratemoiety comprised in said erythropoietin.
 26. A pharmaceuticalcomposition comprising, in a therapeutically effective amount, a HESderivative obtainable by a method comprising selectively reacting HES offormula (I)

at its reducing end which is not oxidized prior to said reaction, in anaqueous medium with O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine,wherein R₁, R₂ and R₃ are independently hydrogen or a 2-hydroxyethylgroup, wherein the resulting product is reacted via its unreacted O—NH₂group with at least one further compound, or wherein theO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine is first reacted via oneof its O—NH₂ groups with at least one further compound prior to thereaction with compound (I), and wherein theO-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxyl amine is then reacted via theunreacted O—NH₂ group with compound (I) at its reducing end which is notoxidized, and wherein the at least one further compound is apolypeptide.
 27. A pharmaceutical composition as claimed in claim 26wherein the polypeptide is reacted via an oxidized carbohydrate moietycomprised in the polypeptide.
 28. A pharmaceutical composition asclaimed in claim 27 wherein the polypeptide is erythropoietin.
 29. Apharmaceutical composition as claimed in claim 28 wherein theerythropoietin is oxidized with sodium periodate prior to the reaction.30. A pharmaceutical composition as claimed in claim 28 wherein theerythropoietin is partially desialylated and subsequently oxidized withsodium periodate prior to the reaction.
 31. A pharmaceutical compositioncomprising, in a therapeutically effective amount, a HES derivativeobtainable by a method comprising selectively reacting HES of formula(I)

at its reducing end which is not oxidized prior to said reaction, in anaqueous medium with a compound of formula

wherein R₁, R₂ and R₃ are independently hydrogen or a 2-hydroxyethylgroup, and wherein the reaction product of compound (I) with thecompound of formula

is further reacted via the unreacted —O—NH₂ in an aqueous medium witherythropoietin via an oxidized carbohydrate moiety comprised in saiderythropoietin.
 32. A HES derivative as claimed in claim 1, wherein saidreacting is in a solvent selected from the group consisting of water,dimethylsulfoxide (DMSO), dimethylformamide (DMF), methanol, ethanol,and a mixture of water with one or more of DMSO, DMF, methanol, andethanol.
 33. A HES derivative as claimed in claim 32, wherein saidreacting is in a solvent that is at least 50% water by weight.
 34. A HESderivative as claimed in claim 1, wherein said reacting is at atemperature of 25° C. to 35° C.
 35. A HES derivative as claimed in claim1, wherein the time of said reacting is from two hours to 48 hours. 36.A HES derivative as claimed in claim 1, wherein said reacting is at a pHfrom 4.5 to 6.5.
 37. A HES derivative as claimed in claim 1, whereinsaid reacting is at a temperature of about 25° C. and a pH of about 5.5.